Tissue engineered blood vessel

ABSTRACT

Compositions and methods of using tissue engineered blood vessels to repair and regenerate blood vessels of patients with vascular disease are disclosed.

RELATED APPLICATIONS

This application is a non-provisional filing of a provisionalapplication U.S. Pat. App. No. 61/049,067.

FIELD OF THE INVENTION

The invention relates to tissue engineered blood vessels for treatmentof vascular disease. In particular, the invention provides tissueengineered blood vessels prepared from scaffolds, and one or more ofcells, cell sheets, cell lysate, minced tissue, and bioreactor processesto repair or replace a native blood vessel that has been damaged ordiseased.

BACKGROUND OF THE INVENTION

Cardiovascular-related disorders are a leading cause of death indeveloped countries. In the US alone, one cardiovascular death occursevery 34 seconds and cardiovascular disease-related costs areapproximately $250 billion. Current methods for treatment of vasculardisease include chemotherapeutic regimens, angioplasty, insertion ofstents, reconstructive surgery, bypass grafts, resection of affectedtissues, or amputation. Unfortunately, for many patients, suchinterventions show only limited success, and many patients experience aworsening of the conditions or symptoms.

These diseases often require reconstruction and replacement of bloodvessels. Currently, the most popular source of replacement vessels isautologous arteries and veins. However, such autologous vessels are inshort supply or are not suitable especially in patients who have hadvessel disease or previous surgeries.

Synthetic grafts made of materials such, as PTFE and Dacron are popularvascular substitutes. Despite their popularity, synthetic materials arenot suitable for small diameter grafts or in areas of low blood flow.Material-related problems such as stenosis, thromboembolization, calciumdeposition, and infection have also been demonstrated.

Therefore, there is a clinical need for biocompatible and biodegradablestructural matrices that facilitate tissue infiltration torepair/regenerate diseased or damaged tissue. In general, the clinicalapproaches to repair damaged or diseased blood vessel tissue do notsubstantially restore their original function. Thus, there remains astrong need for alternative approaches for tissue repair/regenerationthat avoid the common problems associated with current clinicalapproaches.

The emergence of tissue engineering may offer alternative approaches torepair and regenerate damaged/diseased tissue. Tissue engineeringstrategies have explored the use of biomaterials in combination withcells, growth factors, bioactives and bioreactor processes to developbiological substitutes that ultimately can restore or improve tissuefunction. The use of colonizable and remodelable scaffolding materialshas been studied extensively as tissue templates, conduits, barriers,and reservoirs. In particular, synthetic and natural materials in theform of foams, and textiles have been used in vitro and in vivo toreconstruct/regenerate biological tissue, as well as deliver agents forinducing tissue growth.

Such tissue-engineered blood vessels (TEBVs) have been successfullyfabricated in vitro and have been used in animal models. However, therehas been very limited clinical success.

Regardless of the composition of the scaffold and the targeted tissue,the template must possess some fundamental characteristics. The scaffoldmust be biocompatible, possess sufficient mechanical properties toresist the physical forces applied at the time of surgery, porous enoughto allow cell invasion, or growth, easily sterilized, able to beremodeled by invading tissue, and degradable as the new tissue is beingformed. Furthermore, the scaffold may be fixed to the surrounding tissuevia mechanical means, fixation devices, or adhesives. So far,conventional materials, alone or in combination, lack one or more of theabove criteria. Accordingly, there is a need for scaffolds that canresolve the potential pitfalls of conventional materials.

SUMMARY OF THE INVENTION

The invention is a tissue engineered blood vessel (TEBV) comprised of abiocompatible, bioabsorbable scaffold and one or more of cells, cellsheets, cell lysate, minced tissue, and cultured with or without abioreactor process. Such tissue engineered blood vessels may be used torepair or replace a native blood vessel that has been damaged ordiseased. In one embodiment, the tissue engineered blood vessel iscomprised of a biocompatible, bioabsorbable scaffold and cells. Inanother embodiment the tissue engineered blood vessel is comprised of abiocompatible, bioabsorbable scaffold and cell sheets. In anotherembodiment the tissue engineered blood vessel is comprised of abiocompatible, bioabsorbable scaffold and cell lysate. In yet anotherembodiment the tissue engineered blood vessel is comprised of abiocompatible, bioabsorbable scaffold and minced tissue. In addition,various combinations of cells, cell sheets, cell lysate and mincedtissue are combined with a biocompatible, bioabsorbable scaffold to formthe tissue engineered bloods vessel. These tissue engineered bloodvessels may be cultured with or without a bioreactor process. In oneembodiment, the tissue engineered blood vessel is enhanced by combiningwith bioactive agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Human umbilical cord—derived cell (hUTC) attachment and growthon a vascular graft biomaterial after 3 and 7 days in culture. A:PDO-ESS scaffold after 3 days, B: PDO-ESS scaffold after 7 days, C:PDO/collagen-ESS scaffold after 3 days, D: PDO/collagen-ESS scaffoldafter 7 days. All images taken at 40× magnification

FIG. 2. Representative example of smooth muscle cell (UASMC) cellattachment and growth on a vascular graft biomaterial after 3 and 7 daysin culture. A: 100 mg/ml PDO-ESS scaffold after 3 days, B: 100 mg/mlPDO-ESS scaffold after 7 days, C: 140 mg/ml PDO-ESS scaffold after 3days, D: 140 mg/ml PDO-ESS scaffold after 7 days. All images taken at40× magnification.

FIG. 3. Representative example of endothelial cell (HUVEC) cellattachment and growth on a vascular graft biomaterial after 3 and 7 daysin culture. A: 100 mg/ml PDO-ESS scaffold after 3 days, B: 100 mg/mlPDO-ESS scaffold after 7 days, C: 140 mg/ml PDO-ESS scaffold after 3days, D: 140 mg/ml PDO-ESS scaffold after 7 days. All images taken at40× magnification.

FIG. 4. Cell attachment to lysate-augmented PDO-ESS scaffolds. PDO-ESSscaffolds were loaded with hUTC cell lysate at two differentconcentrations and lyophilized. Cells (50,000/scaffold) were then seededonto the scaffolds and cultured for 3 and 7 days before Live/Deadstaining (A) control scaffold after 3 days (B) control scaffold after 7days (C) 18 mg/ml lysate-augmented scaffold after 3 days (D) 18 mg/mllysate-augmented scaffold after 7 days (E) 5.2 mg/ml lysate augmentedscaffold after 3 days (F) 5.2 mg/ml lysate-augmented scaffold after 7days. All images taken at 40× magnification.

FIG. 5. DNA content of cells cultured on lysate-augmented PDO-ESSscaffolds. PDO-ESS scaffolds were loaded with hUTC cell lysate at twodifferent concentrations (low=5.2 mg/ml, high=18 mg/ml) and lyophilized.Cells (50,000/scaffold) were then seeded onto the scaffolds and culturedfor 3 days before analysis of cellular DNA content. Samples were washed,digested with papain and DNA quantitated using the CyQuant NF assay kit.

FIG. 6. Tubular scaffolds coated with PBS or hUTC lysate which werecultured with rat SMC for 24 hours showed Lysate coated scaffolds showedmore cell attachment from LIVE/DEAD and H&E image

FIG. 7. H&E staining for the hUTC seeded PDO sheet pre-coated with hUTClysate after cultured 11 days after seeding. While cells were onlyseeded on one side, cells migrated and penetrated all over the scaffold.Box?

FIG. 8. The rolled tube cartoon shows the sampling areas for Live-Deadstaining experiment. In a static culture, position 1 c 2 is the bottomof the tube, a and b are the side of the tube and 1 d 2 the top of thetube.

FIG. 9. H&E staining for the rolled PDO tube from an rSMC seeded sheet(50 micron in thickness).

FIG. 10. Seeding of hUTC on 2 mm tubular scaffolds. PDO-ESS (A) andPDO-ESS coated with rat tail type I collagen (B) tubular scaffolds 2 mmin diameter were secured to LumeGen bioreactor chambers and seeded byfilling the lumen with a cell suspension at 5.5×10⁵ cells/ml. Thechambers were then rotated at 0.4 rpm overnight and analyzed byLive/Dead staining All images taken at 100× magnification.

FIG. 11. Seeding of rat aortic smooth muscle cells on tubular scaffolds.PDO-ESS (A) and PDO/collagen-ESS (B) tubular scaffolds were secured toLumeGen bioreactor chambers and seeded by filling the lumen with a cellsuspension at 2×10⁶ cells/ml. The chambers were then rotated at 0.4 rpmovernight and analyzed by Live/Dead staining All images taken at 100×magnification.

FIG. 12. Exposure of rat aortic smooth muscle cells on a tubularscaffold to fluid flow or 7 days of static culture. A PDO-ESS tubularscaffolds ˜5 cm long were secured to LumeGen bioreactor chambers andseeded by filling the lumen with a cell suspension at 2×10⁶ cells/ml.The chambers were then rotated at 0.4 rpm overnight. The next day, thetubular scaffolds were connected to the LumeGen bioreactor and exposedto flow at a rate of 10 ml/min for 2 hours. One tubular scaffold wasthen analyzed by Live/Dead staining (A) Left side of the tube. (B) Rightside of the tube. The other tubular scaffold was incubated staticallyfor an additional 7 days then analyzed by Live/Dead staining (C) Leftside of the tube. (D) Right side of the tube. All images taken at 100×magnification.

FIG. 13. Exposure of rat aortic smooth muscle cells on a tubularscaffold to dynamic or static culture conditions. PDO-ESS tubularscaffolds ˜5 cm long were secured to LumeGen bioreactor chambers andseeded by filling the lumen with a cell suspension at 2×10⁶ cells/ml.The chambers were then rotated at 0.4 rpm overnight. The next day, thetubular scaffolds were connected to the LumeGen bioreactor and exposedto flow at a rate of 20 ml/min and pulsatile pressure ranging from120-80 mm Hg at a frequency of 1 Hz for 3 days. Another tubular scaffoldwas cultured under static conditions for the same time period. Bothtubes were then analyzed by Live/Dead staining (A) Left side of the tubecultured statically. (B) Right side of the tube cultured statically. (C)Left side of the tube cultured under dynamic conditions. (D) Right sideof the tube cultured under dynamic conditions. All images taken at 100×magnification.

FIG. 14. Image showed minced rat muscle tissue were distributeduniformly over tubular constructs with different amount of tissue

FIG. 15. After 72 hours culturing, image showed minced tissue stillattached to the tubular scaffolds

FIG. 16. Rat Smooth Muscle Cells seeded (static) on PDO tubes for 4 daysfollowed by 10 days in bioreactor (H&E staining)

DETAILED DESCRIPTION OF INVENTION

The invention is a tissue engineered blood vessel (TEBV) comprised of abiocompatible, bioabsorbable scaffold and one or more of cells, cellsheets, cell lysate, minced tissue, and cultured with or without abioreactor process. Such tissue engineered blood vessels may be used torepair or replace a native blood vessel that has been damaged ordiseased. In tissue engineering, the rate of resorption of the scaffoldby the body preferably approximates the rate of replacement of thescaffold by tissue. That is to say, the rate of resorption of thescaffold relative to the rate of replacement of the scaffold by tissuemust be such that the structural integrity, e.g. strength, required ofthe scaffold is maintained for the required period of time. If thescaffold degrades and is absorbed unacceptably faster than the scaffoldis replaced by tissue growing therein, the scaffold may exhibit a lossof strength and failure of the device may occur. Additional surgery thenmay be required to remove the failed scaffold and to repair damagedtissue. Thus, devices of the present invention advantageously balancethe properties of biodegradability, resorption, structural integrityover time and the ability to facilitate tissue in-growth, each of whichis desirable, useful or necessary in tissue regeneration or repair. Suchdevices provide synergistic improvements over devices of the prior art.

In general, a suitable biodegradable polymer for preparing the scaffoldis desirably configured so that it has mechanical properties that aresuitable for the intended application, remains sufficiently intact untiltissue has in-grown and healed, does not invoke a minimal inflammatoryresponse or toxic response, is capable of withstanding long-termhemodynamic stress without material failure, resistant to boththrombosis and infection and is metabolized in the body after fulfillingits purpose, is easily processed into the desired final product to beformed, demonstrates acceptable shelf-life, and is easily sterilized.

The biocompatible, biodegradable scaffold may be comprised of natural,modified natural or synthetic biodegradable polymers, includinghomopolymers, copolymers and block polymers, linear or branched,segmented or random; as well as combinations thereof. Particularly wellsuited synthetic biodegradable polymers are aliphatic polyesters whichinclude but are not limited to homopolymers and copolymers of lactide(which includes lactic acid D-,L- and meso lactide), glycolide(including glycolic acid), epsilon-caprolactone, p-dioxanone(1,4-dioxan-2-one), and trimethylene carbonate (1,3-dioxan-2-one). Inone embodiment the polymers are poly(p-dioxanone),poly(lactide-co-glycolide) (PLA/PGA) copolymers (95/5, 85/15, 10/90mole-mole %), and poly(glycolide-co-caprolatone) (PGA/PCL) 65/35copolymers, and poly(lactide-co-caprolatone) (PLA/PCL) (60/40 mole-mole%) copolymers.

Suitable natural polymers include, but are not limited to collagen,atelocollagen, elastic, and fibrin and combinations thereof. In oneembodiment, the natural polymer is collagen. In yet another embodiment,the combination of natural polymer is a acellular omental matrix.

The scaffold has dimensions that reflect desired ranges that, incombination with the one or more of cells, cell sheets, cell lysate,minced tissue, and a bioreactor process will replace a small diameter,damaged or diseased vein or artery blood vessel. Desirable dimensionsinclude but are not limited to: internal diameter (3-7 mm preferable,4-6 mm most preferable); wall thickness (0.1-1 mm preferable, 0.2-0.7 mmmost preferable); and length (1-20 cm preferable, 2-10 cm mostpreferable). The table below shows how the properties of our PDOconstruct align with those of a natural vessel.

Dimensions Physical Properties Wall Burst Suture Tensile ID ThicknessLength Compliance Pressure retention (peak (mm) (mm) (cm) (%) (mmHg)(gmf) stress) PDO 2 & 5 0.5 1-20 0.5-1  1500-2500 310   5 MPa Vessel 2 &5 0.5-0.7 1-20 0.2-10 1500-4500 100-500 2-20 MPa

The scaffold has physical properties that reflect desired ranges that,in conjunction with one or more of cells, cell sheets, cell lysate,minced tissue tissue, and a bioreactor process will replace a smalldiameter, damaged or diseased vein or artery blood vessel. Desirablephysical properties include but are not limited to: compliance (0.2-10%preferable, 0.7-7% most preferable); suture retention strength (100 gm-4Kg preferable, 100-300 gm most preferable); burst strength/pressure(1000-4500 mm Hg preferable, 1500-4500 mm Hg most preferable withgreater than 100 mmHg during the bioreactor process); kink resistance(resist kinking during handling during all stages of process-cellseeding, bioreactor, implantation, life of patient); and in-vitrostrength retention (1 day-1 yr maintain enough strength until cell andECM growth overcomes physical property losses of scaffold; 1 day-3 mosunder bioreactor “flow” conditions preferable). The scaffold should alsohave desirable tensile properties (radial and axial) that include butare not limited to: elastic modulus (MPa) of longitudinal/axial (1-200preferable; 5-100 most preferable) and orthogonal/radial (0.1-100preferable, 0.5-50 most preferable) and random (0.1-100 preferable,0.5-50 most preferable) and wet/longitudinal (5-100 preferable, 25-75preferable); a peak stress (MPa) of longitudinal/axial (1-30 preferable;2-20 most preferable) and orthogonal/radial (0.5-15n preferable, 1-10most preferable) and random (0.5-15 preferable, 1-10 most preferable)and wet/long (1-30 preferable; 2-20 most preferable); failure strain (%)of longitudinal/axial (1-200 preferable; 5-75 most preferable) andorthogonal/radial (5-400 preferable, 10-300 most preferable) and random(5-400 preferable, 10-300 most preferable) and wet/long (1-200preferable; 20-100 most preferable).

The scaffold has morphology that reflects desired ranges that, inconjunction with one or more of cells, cell sheets, cell lysate, mincedtissue, and a bioreactor process will replace a small diameter, damagedor diseased vein or artery blood vessel. Desirable morphology includesbut is not limited to: pore size (1-200 um preferable, most preferableless than 100 um); porosity (40-98% preferable, most preferable 60-95%);surface area/vol (0.1-7 m²/cm³ preferable, most preferable 0.3-5.5m²/cm³); water permeability (1-10 ml cm²/min @80-120 mmHg preferable,most preferable <5 ml cm²/min @120 mmHg); and orientation ofpolymer/fibers (allows proper cell seeding, adherence, growth, and ECMformation). Polymer/fiber orientation will also allow proper cellmigration; also important for the minced tissue fragments such thatcells will migrate out of the fragments and populate the scaffold.

The scaffold has biocompatibility that reflects desired properties for ascaffold that, in conjunction with one or more of cells, cell sheets,cell lysate, minced tissue and a bioreactor process will replace a smalldiameter, damaged or diseased vein or artery blood vessel. Desirablebiocompatibility includes but is not limited to: absorption (1 wk-4 yrspreferable, most preferable 4 wks-30 wks to allow greatest vol ofscaffold to be occupied by cells and ECM); tissue reaction (minimal);cell compatibility (adherence, viability, growth, migration anddifferentiation not negatively impacted by scaffold); residual solvent(minimal); residual EtO (minimal); and hemocompatible(non-thrombogenic).

The scaffold has other factors that reflect desired properties for ascaffold that, in conjunction with one or more of cells, cell sheets,cell lysate, minced tissue, tissue and a bioreactor process will replacea small diameter, damaged or diseased vein or artery blood vessel.Desirable factors includes but are not limited to: surface energy(allows proper cell seeding, adherence, growth, migration and ECMformation); surface chemistry (addition of factors such as oxygen,surface roughness or topography (can be utilized to affect cellattachment and other cell functions), nanoscale features on the surfacepreferably in a size range of 10-1200 nanometers; more preferably 25-900nanometers (allow preferential endothelial cell attachment), NO,free-radical scavengers allows proper cell seeding, adherence, growth,and ECM formation); cell mediators (addition of factors such as matrixproteins allows proper cell seeding, adherence, growth, migration andECM formation); hydrophobicity/hydrophilicity (proper balance ofhydrophobicity/hydrophilicity allows proper cell seeding, adherence,growth, and ECM formation). Other surface modifications includeproviding electrical microcurrent in a form of coating a surface withgalvanic materials. In particular, zinc and copper (0.01 microns-0.1microns) can act as an electrical current source enhancing endothelialand smooth muscle cell attachment and proliferation.

Non-limiting examples of a scaffold that may be used in the presentinvention include textile structures such as felts, weaves, knits,braids, meshes, non-wovens, warped knits; foams, including porous foamsand semi-porous foams; perforated films or sheets; patterned films orsheets or fibers; and combinations thereof. As used herein, the term“nonwoven fabric” includes, but is not limited to, bonded fabrics,formed fabrics, or engineered fabrics, that are manufactured byprocesses other than spinning, weaving or knitting. More specifically,the term “nonwoven fabric” refers to a porous, textile-like material,usually in flat sheet form, composed primarily or entirely of staplefibers assembled in a web, sheet or batt. The fiber diameter ispreferably 10 nm to 100 um and more preferably 25 nm to 10 um. In oneembodiment, the scaffold is a textile, a foam and combinations thereof.

In another embodiment, the scaffold is a textile comprised of fibersprepared by electrostatic spinning, extrusion, injection molding, aswell as any pre- or post-processes (ex. laser cutting to form pours inextruded tube). In one embodiment, the scaffold is a textile prepared byelectrostatic spinning. In the electrostatically spun scaffold process,an electrical force is applied to the polymeric solution that overcomesthe surface tension of the solution, forming a charged jet. This jet ofsolution is ejected, dried and solidified onto a substrate to form asheet, tube or other construct comprised of the electrostatically spunfibers. Spinnability of the polymeric solution is controlled by severalparameters that include but are not limited to: concentration (aconcentration that allows polymer/solvent solution to be spun and yieldfibers that form a proper scaffold (1-50 w/v % preferable); solvent (asolvent that dissolves the polymer in the given concentration range,HFIP preferable); solution viscosity (10-300 mg/ml preferable, 25-250mg/ml most preferable (50-3000 centipoise)). By controlling the spinningconditions, the resulting fibers can range from about 0.1 μm to about 10μm and preferably will range from about 0.3 μm to about 5.0 μm.

Other processing parameters for electrostatic spinning that areimportant include but are not limited to: voltage potential (10-100 kVpreferable, most preferable 15-30 kV); flow rate (0.1-20 ml/hrpreferable, 1-15 ml/hr most preferable); gap/tip distance (1-35 cmpreferable, 2.5-25 cm most preferable); rotation/mandrel rate/speed(10-5,000 rpm preferable, 50-3000 rpm most preferable). The fibers canalso be spun from the melt.

Optionally, the strength of the electrostatically spun scaffold (ESSC)may be improved by bonding the fibers of the aforementioned construct.Bonding of the fibers may be accomplished by coating the ESSC with a lowmelting materials such as PCL and low molecular PLGA copolymers. Aftercoating the scaffold, a post process using a heat press may beperformed. The process melts the coated layer on the reinforcementfibers. The molten coating between the electrospun fibers is compressedand fuses the fibers together upon cooling to room temperature.Alternatively, during the electrospinning process, electrospun fibersare exposed with the vapor of the solvent. Upon curing, the fibers fusetogether thereby strengthening the scaffold.

In one embodiment the textiles and ESSC scaffolds are prepared frompolymers including, but not limited to are poly(p-dioxanone),poly(lactide-co-glycolide) (PLA/PGA) copolymers (95/5, 85/15, 10/90mole-mole %), and collagen.

In another embodiment the scaffold is a foam. In one embodiment, thefoam scaffolds are prepared from elastomeric copolymers. Suitablebioabsorbable, biocompatible elastomeric copolymers include but are notlimited to copolymers of epsilon-caprolactone and glycolide (preferablyhaving a mole ratio of epsilon-caprolactone to glycolide of from about30:70 to about 70:30, preferably 35:65 to about 65:35, and morepreferably 45:55 to 35:65); elastomeric copolymers ofepsilon-caprolactone and lactide, including L-lactide, D-lactide blendsthereof or lactic acid copolymers (preferably having a mole ratio ofepsilon-caprolactone to lactide of from about 35:65 to about 65:35 andmore preferably 45:55 to 30:70) elastomeric copolymers of p-dioxanone(1,4-dioxan-2-one) and lactide including L-lactide, D-lactide and lacticacid (preferably having a mole ratio of p-dioxanone to lactide of fromabout 40:60 to about 60:40); elastomeric copolymers ofepsilon-caprolactone and p-dioxanone (preferably having a mole ratio ofepsilon-caprolactone to p-dioxanone of from about 30:70 to about 70:30);elastomeric copolymers of p-dioxanone and trimethylene carbonate(preferably having a mole ratio of p-dioxanone to trimethylene carbonateof from about 30:70 to about 70:30); elastomeric copolymers oftrimethylene carbonate and glycolide (preferably having a mole ratio oftrimethylene carbonate to glycolide of from about 30:70 to about 70:30);elastomeric copolymer of trimethylene carbonate and lactide includingL-lactide, D-lactide, blends thereof or lactic acid copolymers(preferably having a mole ratio of trimethylene carbonate to lactide offrom about 30:70 to about 70:30) and blends thereof. In one embodiment,the elastomeric copolymer is poly(glycolide-co-caprolatone) (PGA/PCL)65/35 copolymer or a poly(lactide-co-caprolatone) (PLA/PCL) (60/−40mole-mole %) copolymer.

Foam scaffolds may be prepared by conventional processes such as,lyophilization, supercritical solvent foaming (i.e., as described in EP464,163 B1), gas injection extrusion, gas injection molding or castingwith an extractable material In one embodiment, the foams are preparedby lyophilization. Suitable methods for lyophilizing elastomericpolymers such as 65/35 PGA/PCL to form foams is described in theexamples of U.S. Pat. No. 6,355,699, “Process for ManufacturingBiomedical Foams”, assigned to Ethicon, Inc incorporated herein byreference in its entirety.

In another embodiment, leachables can be introduced into the scaffold asan additional method to form pores. Suitable leachable solids includenontoxic leachable materials such as salts (e.g., sodium chloride,potassium chloride, calcium chloride, sodium tartrate, sodium citrate,and the like), biocompatible mono and disaccharides (e.g., glucose,fructose, dextrose, maltose, lactose and sucrose), polysaccharides(e.g., starch, alginate, chitosan), water soluble proteins (e.g.,gelatin and agarose).

The foams have microstructures suitable for tissue engineering. Thefeatures of such foams can be controlled to suit a desired applicationby choosing the appropriate conditions to form the foam duringlyophilization. These features in absorbable polymers have distinctadvantages over the prior art where the scaffolds are typicallyisotropic or random structures. However, it is preferred that foams usedin tissue engineering (i.e. repair or regeneration) have a structurethat provides organization at the microstructural level that provides atemplate that facilitates cellular organization and regeneration oftissue that has the anatomical, biomechanical, and biochemical featuresof normal tissues. These foams can be used to repair or regeneratetissue (including organs) in animals such as domestic animals, primatesand humans.

The features of such foams can be controlled to suit desired applicationby selecting the appropriate conditions for lyophilization to obtain oneor more of the following properties: (1) interconnecting pores of sizesranging from about 10 to about 200 μm (or greater) that provide pathwaysfor cellular ingrowth and nutrient diffusion; (2) a variety ofporosities ranging from about 20% to about 98% and preferably rangingfrom about 50% to about 95%; (3) gradient in the pore size across onedirection for preferential cell culturing; (4) channels that run throughthe foam for improved cell invasion, vascularization and nutrientdiffusion (5) micro-patterning of pores on the surface for cellularorganization; (6) tailorability of pore shape and/or orientation (e.g.substantially spherical, ellipsoidal, columnar); (7) anisotropicmechanical properties; (8) composite foams with a polymer compositiongradient to elicit or take advantage of different cell response todifferent materials; (9) blends of different polymer compositions tocreate structures that have portions that will break down at differentrates; (10) foams co-lyophilized or coated with pharmaceutically activecompounds; (11) and the ability to make 3 dimensional shapes and deviceswith preferred microstructures. The inner, or luminal, layer of thescaffold may be optimized for endothelialization through control of theporosity of the surface and the possible addition of a surfacetreatment. The outermost, or adventitial, layer of the scaffold may betailored to support smooth muscle cell growth, again by optimizing theporosity (percent porosity, pore size, pore shape and pore sizedistribution) and by incorporating bioactive factors, pharmaceuticalagents, or cells. There may or may not be a barrier layer with lowporosity disposed between these two porous layers to increase strengthand decrease leakage. Such structural features of the scaffold describedherein can also be found in textiles such as, electrostatically spunscaffolds and other scaffolds described herein.

In one embodiment, the scaffold is a combination of foams and textiles.Textiles include woven, knitted, warped knitted (i.e., lace-like),non-woven, and braided structures that act as a reinforcement for thescaffold. The reinforcement should have a sufficient density to permitsuturing, but the density should not be so great as to impede properbonding between the foam and the textile. The reinforcing material mayalso be formed from a thin, perforation-containing elastomeric sheetwith perforations to allow tissue ingrowth.

For example, the present invention also provides a composite scaffoldcomprising a first layer that is a textile layer and a second layer ofbiocompatible foam or ESSC. This composite structure allows for thecreation of structures with unique mechanical properties. In oneembodiment the textile layer could allow the use of sutures, staples orvarious fixation devices to hold the composite in place. Generally, thetextile has a thickness in the range of about 1 micron to 500 microns.The textile layer allows the composite to have variable mechanicalstrength depending on the design, a different bioabsorption profile, anda different microenvironment for cell invasion and seeding, which areadvantageous in a variety of medical applications. The textile layer maybe made from a variety of biocompatible polymers and blends ofbiocompatible polymers, which are preferably bioabsorbable. Thebiocompatible foam or ESSC may be either contain gradients or channels.The gradient structure has a substantially continuous transition in atleast one characteristic selected from the group consisting ofcomposition, stiffness, flexibility, bioabsorption rate, porearchitecture and/or microstructure. The gradient structure can be madefrom a blend of absorbable polymers that form compositional gradienttransitions from one polymeric material to a second polymeric material.In situations where a single chemical composition is sufficient for theapplication, the invention provides a composite that may havemicrostructural variations in the structure across one or moredimensions that may mimic the anatomical features of the tissue. Thechanneled structure provides channels that extend through the foam tofacilitate cell migration and nutrient flow throughout the channeledstructure.

In another embodiment the foam or ESSC may have a textile fused to thetop or bottom surface. This way, surface properties of the structure canbe controlled such as porosity, permeability, degradation rate andmechanical properties. The textile can be produced via conventionaltechniques, described herein, and in which a textile can be built up ona lyophilized foam surface. The textile can be produced via anelectrostatic spinning process in which a ESSC can be built up on alyophilized foam surface.

The scaffold may include one or more layers of each of the foam or ESSCand reinforcement components. Preferably, adjacent layers of foam orESSC are also integrated by at least a partial interlocking of thepore-forming webs or walls in the adjacent layers.

In one embodiment the scaffold may be coated with natural polymers toenhance cellular compatibility. Suitable natural polymers include, butare not limited to collagen, atelocollagen, elastin, hyaluronic acid andfibrin and combinations thereof. In one embodiment, the natural polymeris collagen. In yet another embodiment, the combination of naturalpolymer is acellular omental matrix.

In one embodiment, the tissue engineered blood vessel is comprised of abiocompatible, bioabsorbable scaffold and cells. The scaffold is asdescribed herein above. Suitable cells that may be combined with thescaffold include, but are not limited to, stem cells such as multipotentor pluripotent stem cells; progenitor cells, such as smooth muscleprogenitor cells and vascular endothelium progenitor cells; embryonicstem cells; postpartum tissue derived cells such as, placental tissuederived cells and umbilical tissue derived cells, endothelial cells,such as vascular endothelial cells; smooth muscle cells, such asvascular smooth muscle cells; precursor cells derived from adiposetissue; and arterial cells such as, cells derived from the radial arteryand the left and right internal mammary artery (IMA), also known as theinternal thoracic artery.

In one embodiment, the TEBV comprises a scaffold as described hereinabove and human umbilical tissue derived cells (hUTCs). The methods forisolating and collecting human umbilical tissue-derived cells (hUTCs)(also referred to as umbilical-derived cells (UDCs)) are described incopending U.S. application Ser. No. 10/877,012 incorporated herein byreference in its entirety. In another embodiment, the TEBV comprises ascaffold as described herein above, human umbilical tissue derived cells(hUTCs) and one or more other cells. The one or more other cellsincludes, but is not limited to vascular smooth muscle cells (SMCs),vascular smooth muscle progenitor cells, vascular endothelial cells(ECs), or vascular endothelium progenitor cells, and/or othermultipotent or pluripotent stem cells. hUTCs in combination with one ormore other cells on the scaffold may enhance the seeding, attachment,and proliferation of for example ECs and SMCs on the scaffold. hUTCS mayalso promote the differentiation of the EC or SMC or progenitor cells inthe scaffold construct. This may promote the maturation of TEBVs duringthe in vitro culture as well as the engraftment during the in vivoimplantation. hUTCs may provide trophic support, or provide and enhancethe expression of ECM proteins. The trophic effects of the cells,including hUTCs, can lead to proliferation of the vascular smooth muscleor vascular endothelium of the patient. The trophic effects of thecells, including hUTCs, may induce migration of vascular smooth musclecells, vascular endothelial cells, skeletal muscle progenitor cells,vascular smooth muscle progenitor cells, or vascular endotheliumprogenitor cells to the site or sites of the regenerated blood vessel.

Cells can be harvested from a patient (before or during surgery torepair the tissue) and the cells can be processed under sterileconditions to provide a specific cell type. One of skill in the art isaware of conventional methods for harvesting and providing the cells asdescribe above such as described in Osteoarthritis Cartilage 2007February; 15(2):226-31 and incorporated herein by reference in theirentirety.

The cells can be seeded on the scaffolds of the present invention for ashort period of time, e.g. less than one day, just prior toimplantation, or cultured for longer a period, e.g. greater than oneday, to allow for cell proliferation and extracellular matrix synthesiswithin the seeded scaffold prior to implantation. In one embodiment, asingle cell type is seeded on the scaffold. In another embodiment, oneor more cell types are seeded on the scaffold. Various cellularstrategies could be used with these scaffolds (i.e., autologous,allogenic, xenogeneic cells etc.).

In another embodiment the cells are genetically modified to expressgenes of interest responsible for pro-angiogenic activity,anti-inflammatory activity, cell survival, cell proliferation ordifferentiation or immunemodulation.

In another embodiment the tissue engineered blood vessel is comprised ofa biocompatible, bioabsorbable scaffold and cell sheets. Cell sheets maybe made of hUTCs or other cell types. Methods of making cell sheets areas described in copending U.S. application Ser. No. 11/304,091incorporated herein by reference in its entirety. The cell sheet isgenerated using thermoresponsive polymer coated dishes that allowharvesting intact cell sheets with the decrease of the temperature.Alternatively, other methods of making cell sheets include, but are notlimited to growing cells in a form of cell sheets on a polymer film.Selected cells may be cultured on a surface of glass, ceramic or asurface-treated synthetic polymer. For example, polystyrene that hasbeen subjected to a surface treatment, like gamma-ray irradiation orsilicon coating, may be used as a surface for cell culture. Cells grownto over 85% confluence form cell sheet layer on cell growth supportdevice. Cell sheet layer may be separated from cell growth supportdevice using proteolysis enzymes, such as trypsin or dispase.Non-enzymatic cell dissociation could also be used. A non-limitingexample includes a mixture of chelators sold under the trade nameCELLSTRIPPER (Mediatech, Inc., Herndon, Va.), a non-enzymatic celldissociation solution designed to gently dislodge adherent cells inculture while reducing the risk of damage associated with enzymatictreatments.

Alternatively, the surface of the cell growth support device, from whichcultured cells are collected, may be a bed made of a material from whichcells detach without a proteolysis enzyme or chemical material. The bedmaterial may comprise a support and a coating thereon, wherein thecoating is formed from a polymer or copolymer which has a criticalsolution temperature to water within the range of 0° C. to 80° C.

In one embodiment, one or more cells sheets are combined with thescaffold as described herein above by wrapping the cell sheet or sheetsaround the scaffold. The one or more cell sheets may be of the same celltype or of different cell types as described herein above. In oneembodiment, multiple cell sheets could be combined to form a robustvascular construct. For example, cell sheets made of endothelial cellsand smooth muscle cells could be combined with the scaffold to formTEBVs. Alternatively, other cell types such as hUTC cell sheets could becombined with endothelial cell sheets and the scaffold to form TEBV.Furthermore, cell sheets made of hUTCs can be wrapped around apre-formed TEBV composed of a scaffold, ECs, and SMCs to provide trophicfactors supporting maturation of the construct.

Cell sheets may be grown on the scaffold to provide reinforcement andmechanical properties to the cell sheets. Reinforced cell sheets can beformed by placing biodegradable or non-biodegradable reinforcing membersat the bottom of support device prior to seeding support device withcells. Reinforcing members are as described herein above. Cell sheetlayer that results will have incorporated the reinforcing scaffoldproviding additional strength to cell sheet layer, which can bemanipulated without the requirement for a backing layer. A preferredreinforcing scaffold is a mesh comprised of poly(dioxanone). The meshcan be placed at the bottom of a Corning® Ultra low attachment dish.Cells can then be seeded on to the dishes such that they will formcell-cell interactions but also bind to the mesh when they interact withthe mesh. This will give rise to reinforced cell sheets with betterstrength and handling characteristics. Such reinforced cell sheets maybe rolled into TEBV or the reinforced cell sheet layer may be disposedon a scaffold (as described above).

In another embodiment, the cell sheet is genetically engineered. Thegenetically engineered cell sheet comprises a population of cellswherein at least one cell of the population of cells is transfected withan exogenous polynucleotide such that the exogenous polynucleotideexpresses express diagnostic and/or therapeutic product (e.g., apolypeptide or polynucleotide) to assist in tissue healing, replacement,maintenance and diagnosis. Examples of “proteins of interest” (and thegenes encoding same) that may be employed herein include, withoutlimitation, cytokines, growth factors, chemokines, chemotactic peptides,tissue inhibitors of metalloproteinases, hormones, angiogenesismodulators either stimulatory or inhibitory, immune modulatory proteins,neuroprotective and neuroregenerative proteins and apoptosis inhibitors.More specifically, preferred proteins include, without limitation,erythropoietin (EPO), EGF, VEGF, FGF, PDGF, IGF, KGF, IFN-α, IFN-δ, MSH,TGF-α, TGF-β, TNF-α, IL-1, BDNF, GDF-5, BMP-7 and IL-6.

In another embodiment the tissue engineered blood vessel is comprised ofa biocompatible, bioabsorbable scaffold and cell lysate. Cell lysatesmay be obtained from cells including, but not limited to stem cells suchas multipotent or pluripotent stem cells; progenitor cells, such assmooth muscle progenitor cells and vascular endothelium progenitorcells; embryonic stem cells; postpartum tissue derived cells such as,placental tissue derived cells and umbilical tissue derived cells,endothelial cells, such as vascular endothelial cells; smooth musclecells, such as vascular smooth muscle cells; precursor cells derivedfrom adipose tissue; and arterial cells such as, cells derived from theradial artery and the left and right internal mammary artery (IMA), alsoknown as the internal thoracic artery. The cell lysates and cell solublefractions may be stimulated to differentiate along a vascular smoothmuscle or vascular endothelium pathway. Such lysates and fractionsthereof have many utilities. Use of lysate soluble fractions (i.e.,substantially free of membranes) in vivo, for example, allows thebeneficial intracellular milieu to be used allogenically in a patientwithout introducing an appreciable amount of the cell surface proteinsmost likely to trigger rejection, or other adverse immunologicalresponses. Methods of lysing cells are well-known in the art and includevarious means of mechanical disruption, enzymatic disruption, orchemical disruption, or combinations thereof. Such cell lysates may beprepared from cells directly in their growth medium and thus containingsecreted growth factors and the like, or may be prepared from cellswashed free of medium in, for example, PBS or other solution.

In yet another embodiment the tissue engineered blood vessel iscomprised of a biocompatible, bioabsorbable scaffold and minced tissue.Minced tissue has at least one viable cell that can migrate from thetissue fragments onto the scaffold. More preferably, the minced tissuecontains an effective amount of cells that can migrate from the tissuefragments and begin populating the scaffold. Minced tissue may beobtained from one or more tissue sources or may be obtained from onesource. Minced tissue sources include, but are not limited to muscletissue, such as skeletal muscle tissue and smooth muscle tissue;vascular tissue, such as venous tissue and arterial tissue; skin tissue,such as endothelial tissue; and fat tissue.

The minced tissue is prepared by first obtaining a tissue sample from adonor (autologous, allogeneic, or xenogeneic) using appropriateharvesting tools. The tissue sample is then finely minced and dividedinto small fragments either as the tissue is collected, oralternatively, the tissue sample can be minced after it is harvested andcollected outside the body. In embodiments where the tissue sample isminced after it is harvested, the tissue samples can be washed threetimes in phosphate buffered saline. The tissue can then be minced intosmall fragments in the presence of a small quantity, for example, about1 ml, of a physiological buffering solution, such as, phosphate bufferedsaline, or a matrix digesting enzyme, such as 0.2% collagenase in Ham'sF12 medium. The tissue is minced into fragments of approximately 0.1 to1 mm³ in size. Mincing the tissue can be accomplished by a variety ofmethods. In one embodiment, the mincing is accomplished with two sterilescalpels cutting in parallel and opposing directions, and in anotherembodiment, the tissue can be minced by a processing tool thatautomatically divides the tissue into particles of a desired size. Inone embodiment, the minced tissue can be separated from thephysiological fluid and concentrated using any of a variety of methodsknown to those having ordinary skill in the art, such as for example,sieving, sedimenting or centrifuging. In embodiments where the mincedtissue is filtered and concentrated, the suspension of minced tissuepreferably retains a small quantity of fluid in the suspension toprevent the tissue from drying out.

The suspension of minced living tissue can be used to create a TEBVaccording to the present invention by depositing the suspension ofliving tissue upon a biocompatible scaffold, such that the tissue andthe scaffold become associated. Preferably, the tissue is associatedwith at least a portion of the scaffold. The TEBV can be implanted in asubject immediately, or alternatively, the construct can be incubatedunder sterile conditions that are effective to maintain the viability ofthe tissue sample.

In another aspect of the invention, the minced tissue could consist ofthe application of two distinct minced tissue sources (e.g., one surfacecould be loaded with minced endothelial tissue and the other surfacecould be loaded with mince smooth muscle tissue).

In one embodiment, the tissue engineered blood vessels comprising ascaffold and one or more of cells, cell sheets, cell lysate, or mincedtissue is enhanced by combining with bioactive agents. Suitablebioactive agents include, but are not limited to an antithrombogenicagent, an anti-inflammatory agent, an immunosuppressive agent, animmunomodulatory agent, pro-angiogenic, an antiapoptotic agent,antioxidants, growth factors, angiogenic factors, myoregenerative ormyoprotective drugs, conditioned medium, extracellular matrix proteins,such as, collagen, atelocollagen, laminin, fibronectin, vitronectin,tenascin, integrins, glycosaminoglycans (hyaluronic acid, chondroitinsulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate andthe like), elastin and fibrin; growth factors and/or cytokines, such asvascular endothelial cell growth factors, platelet derived growthfactors, epidermal growth factors, fibroblast growth factors, hepatocytegrowth factors, insulin-like growth factors, and transforming growthfactors.

Conditioned medium from cells as described previously herein allows thebeneficial trophic factors secreted by the cells to be usedallogeneically in a patient without introducing intact cells that couldtrigger rejection, or other adverse immunological responses. Conditionedmedium is prepared by culturing cells in a culture medium, then removingthe cells from the medium. Conditioned medium prepared from populationsof cells, including hUTCs, may be used as is, further concentrated, forexample, by ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically-acceptable carriers or diluentsas are known in the art, or combined with other bioactive agentsConditioned medium may be used in vitro or in vivo, alone or combinedwith autologous or allogenic live cells, for example. The conditionedmedium, if introduced in vivo, may be introduced locally at a site oftreatment, or remotely to provide needed cellular growth or trophicfactors to a patient. This same medium may also be used for thematuration of the TEBVs. Alternatively, hUTC or other cells conditionedmedium may also be lyophilized onto the scaffolds prior to seeding withboth ECs and SMCs.

From a manufacturing perspective, hUTCs or other cells, or conditionedmedium may shorten the time for the in vitro culture or fabrication ofTEBVs. This will also result in the use of less starting cells makingautologous sources of ECs and SMCs a more viable option.

In one embodiment, the tissue engineered blood vessels comprising ascaffold and one or more of cells, cell sheets, cell lysate, or mincedtissue is enhanced by combining with bioactive agents. These tissueengineered blood vessels may be cultured with or without a bioreactorprocess. The TEBV scaffolds may be cultured using various cell culturebioreactors, including but not limited to a spinner flask, a rotatingwall vessel (RWV) bioreactor, a perfusion-based bioreactor orcombination thereof. In one embodiment the cell culture bioreactor is arotating wall vessel (RWV) bioreactor or a perfusion-based bioreactor.The perfusion-based bioreactor will consist of a device for securing theTEBV scaffolds and allow culture medium to flow through the lumen of thescaffolds, and may also allow for seeding and culturing of cells on boththe inner (lumen) and outer surfaces of the scaffolds. The perfusionbioreactors may also have the capability of generating pulsatile flowand various pressures for conditioning of the cell-seeded scaffoldsprior to implantation. pulsatile flow stress during bioreactor process(1-25 dynes/cm² over 1 day-1 yr preferable; more preferably a gradualincrease from 1-25 dynes/cm² over 2-4 wks).

The scaffolds having cells, cell sheets, cell lysate, or minced tissueand optionally bioactive agents may be cultured for longer a period,e.g. greater than one day, to allow for cell proliferation and matrixsynthesis within the scaffold prior to implantation. Cells cell sheets,cell lysate, or minced tissue are applied to the scaffold as describedherein above and transferred to the bioreactor for longer term culture,or more preferably, seeded and cultured within the bioreactor. Multiplebioreactors may be also used sequentially, e.g. one for initial seedingof cells, and another for long-term culture.

The process of seeding and culturing cells with a scaffold using abioreactor may be repeated with multiple cell types sequentially, e.g.smooth muscle cells are seeded and cultured for a period of time,followed by seeding and culture of endothelial cells, or simultaneously(e.g. smooth muscle cells on the outer surface, and endothelial cellswith on the inner surface (lumen) of the scaffolds). The construct mayor may not be cultured for a period of time to promote maturation. Thebioreactor conditions can be controlled as to promote proper maturationof the construct. Following the culture period the construct can beremoved and implanted into a vascular site in an animal or human.

General cell culture conditions include temperatures of 37° C. and 5%CO₂. The cell seeded constructs will be cultured in a physiologicalbuffered salt solution maintained at or near physiological pH. Culturemedia can be supplemented with oxygen to support metabolic respiration.The culture media may be standard formulations or modified to optimallysupport cell growth and maturation in the construct. The culture mediamay contain a buffer, salts, amino acids, glucose, vitamins and othercellular nutrients. The media may also contain growth factors selectedto establish endothelial and smooth muscle cells within the construct.Examples of these may include VEGF, FGF2, angiostatin, endostatin,thrombin and angiotensin II. The culture media may also be perfusedwithin the construct to promote maturation of the construct. This mayinclude flow within the lumen of the vessel at pressures and flow ratesthat may be at or near values that the construct may be exposed to uponimplant.

The media is specific for the cell type being cultured (i.e.,endothelial medium for endothelial cells, and smooth muscle cell mediumfor SMCs). For the perfusion bioreactor especially, there are otherconsiderations taken into account such as but not limited to shearstress (related to flow rate), oxygen tension, and pressure.

The TEBVs can be also be electrically stimulated to enhance theattachment or proliferation of the different cell types. The electricalstimulation can be performed during the culture and expansion of thecells prior to the fabrication of the TEBV, during the maturation phaseof the TEBV, or during implantation. Cells, including hUTCs may also beelectrically stimulated during the production of conditioned medium.

The present invention also provides a method for the repair orregeneration of tissue inserting the TEBV described above at a locationon the blood vessel in need of repair. These TEBV structures areparticularly useful for the regeneration of tissue between two or moredifferent types of tissues. For a multi-cellular system in the simplestcase, one cell type could be present on one side of the scaffold and asecond cell type on the other side of the scaffold. Examples of suchregeneration can be (a) vascular tissue with smooth muscle on theoutside and endothelial cells on the inside to regenerate vascularstructures.

The invention also relates to methods of treating tissue using the TEBVprepared by the methods described herein. The TEBV can be used inarteriovenous grafting, coronary artery grafting or peripheral arterygrafting. For example, in a typical AV (arteriovenous) surgicalprocedure used for the treatment of end-stage renal failure patients,the surgeon makes an incision through the skin and muscle of theforearm. An artery and a vein are selected (usually the radial arteryand the cephalic vein) and an incision is made into each. The TEBV isthen used to anastomos the ends of the artery and the vein. The muscleand skin are then closed. After the graft has properly healed (4-6weeks), the successful by-pass can be used to treat the patients blood.

In a coronary by-pass (CABG) procedure, a TEBV would be used forpatients suffering from arteriosclerosis, a common arterial disordercharacterized by arterial walls that have thickened, have lostelasticity, and have calcified. This leads to a decrease in blood supplywhich can lead to damage to the heart, stroke and heart attackes. In atypical CABG procedure, the surgeon opens the chest via a sternotomy.The heart's functions are taken over by a Heart and Lung machine. Thediseased artery is located and one end of the TEBV is sewn onto thecoronary arteries beyond the blockages and the other end is attached tothe aorta. The heart is restarted, the sternum is wired together and theincisions are sutured closed. Within a few weeks, the successful by-passprocedure is fully healed and the patient is functioning normally.

The following examples are illustrative of the principles and practiceof this invention, although not limited thereto. Numerous additionalembodiments within the scope and spirit of the invention will becomeapparent to those skilled in the art once having the benefit of thisdisclosure.

EXAMPLES Example 1 Tubular Scaffolds of Bioabsorbable PolymersFabricated by Electrospinning Processes PDO Electrospun Tubes

1) From High Concentration (140 mg/ml)

Solutions of 140 mg/mL of poly(p-dioxanone) (PDO) (Ethicon, Inc.,Somerville, N.J.) were made with 1,1,1,3,3,3-hexafluoro-2-propanol (HFP,TCI America Inc., Portland, Oreg.) solvent. Solutions were left in a box(dark environment) overnight on a shaker plate to ensure that all PDOhad dissolved and formed a homogenous solution. 4 mL of polymer solutionwere then drawn into a plastic syringe (5 ml) (Beckton Dickinson,Franklin Lakes, N.J.) and placed in a syringe pump (KD Scientific Model100, Holliston, Mass.) to be dispensed at a rate of 8 ml/h. A highvoltage power supply (Spellman CZE1000R; Spellman High VoltageElectronics Corporation, Hauppauge, N.Y.)) was used to apply a voltageof +25 kV to a blunt tip 18 gauge needle fixed to the solutioncontaining syringe. Solutions were electrospun onto a 5 mm diametercylindrical grounded mandrel placed 8 inches from the needle tip androtating at a rate of ˜400 rpm to produce a scaffold of randomlyoriented fibers. Mandrel translation distance was 18 cm, with atranslational speed of 18 cm/s to ensure even coverage along the lengthof the mandrel. Immediately after electrospinning, the mandrel andscaffold were quickly immersed in an ethanol bath, and the scaffold wascarefully slid off the mandrel. The tube (inner diameter: 5 mm,thickness: ˜500 microns, length: 10 cm) was then placed in a fume hoodfor 30 minutes to allow for the evaporation of any residual ethanol.

2) From Medium Concentration (100 mg/ml)

Solutions of 100 mg/ml of PDO were made by placing the polymer in HFPsolvent and leaving the solution overnight in the dark on a shaker plateto ensure that all PDO was dissolved and forms a homogenous solution.The desired volume of polymer solution is then drawn into a plasticBeckton Dickinson syringe and placed in the syringe pump to be dispensedat a rate of 10 ml/hr. Two high voltage power supplies were used. Onewas used to apply a voltage of +20 kV to a blunt tip 18 gauge needlefixed to the solution containing syringe, while the other provides −8 kVto a flat metal target 5″ in diameter placed 6″ behind the groundedmandrel (2 or 5 mm in diameter). The grounded mandrel was placed 8″ fromthe needle tip and rotating at a rate of ˜400 rpm to produce a scaffoldof randomly oriented fibers. Mandrel translation distance was 18 cm,with a translational speed of 18 cm/s. For tubular constructs,immediately after electrospinning the mandrel and scaffold were quicklyimmersed in an ethanol bath to assist in sliding the tube off themandrel. Tubes were then placed in a fume hood for 30 minutes to allowfor the evaporation of any residual ethanol.

3) From Low Concentration (60 mg/ml)

Solutions of 60 mg/mL of PDO were made with HFP solvent. Solutions wereleft in a box (dark environment) overnight on a shaker plate to ensurethat all PDO had dissolved and formed a homogenous solution. 15 mL ofpolymer solution were then drawn into a plastic Beckton Dickinsonsyringe (30 ml) and placed in the syringe pump to be dispensed at a rateof 12 ml/h. Two high voltage power supplies were used. One was used toapply a voltage of +22 kV to a blunt tip 18 gauge needle fixed to thesolution containing syringe, while the other provided −10 kV to a flatmetal target placed 6 inches behind the grounded mandrel. Solutions wereelectrospun onto a 5 mm diameter cylindrical grounded mandrel placed 12inches from the needle tip and rotating at a rate of ˜400 rpm to producea scaffold of randomly oriented fibers. Mandrel translation distance was18 cm, with a translational speed of 18 cm/s. Immediately afterelectrospinning, the mandrel and scaffold were quickly immersed in anethanol bath, and the scaffold was carefully slid off the mandrel. Thetube (inner diameter: 5 mm, thickness: ˜500 microns, length: 10 cm) wasthen placed in a fume hood for 30 minutes to allow for the evaporationof any residual ethanol.

85/15 PLGA Electrospun Tubes

1) From High Concentration (120 mg/ml)

Solutions of 120 mg/mL of poly(lactide-co-glycolide) (Purac,Linolnshire, Ill.) having a mole percent ratio of lactide to glycolideof 85/15 (85/15 PLGA) were made with HFP solvent. Solutions were left ina box (dark environment) overnight on a shaker plate to ensure that all85/15 PLGA had dissolved and formed a homogenous solution. 5 mL ofpolymer solution were then drawn into a plastic Beckton Dickinsonsyringe (5 ml) and placed in the syringe pump to be dispensed at a rateof 8 ml/h. Two high voltage power supplies were used. One was used toapply a voltage of +22 kV to a blunt tip 18 gauge needle fixed to thesolution containing syringe, while the other provided −10 kV to a flatmetal target placed 6 inches behind the grounded mandrel. Solutions wereelectrospun onto a 5 mm diameter cylindrical grounded mandrel placed 8inches from the needle tip and rotating at a rate of ˜400 rpm to producea scaffold of randomly oriented fibers. Mandrel translation distance was18 cm, with a translational speed of 18 cm/s. Prior to electrospinning,the mandrel was wrapped with a small section of aluminum foil to aid intube removal. Upon completion of electrospinning, the foil liner wasslid off the mandrel, and carefully removed from the inside of the tube(inner diameter: 5 mm, thickness: ˜500 microns, length: 10 cm).

2) From Low Concentration (50 mg/ml)

Solutions of 50 mg/mL of 85/15 PLGA were made with HFP solvent.Solutions were left in a box (dark environment) overnight on a shakerplate to ensure that all 85/15 PLGA had dissolved and formed ahomogenous solution. 15 mL of polymer solution were then drawn into aplastic Beckton Dickinson syringe (30 ml) and placed in the syringe pumpto be dispensed at a rate of 12 ml/h. Two high voltage power supplieswere used. One was used to apply a voltage of +22 kV to a blunt tip 18gauge needle fixed to the solution containing syringe, while the otherprovided −5 kV to a flat metal target placed 6 inches behind thegrounded mandrel. Solutions were electrospun onto a 5 mm diametercylindrical grounded mandrel placed 8 inches from the needle tip androtating at a rate of ˜400 rpm to produce a scaffold of randomlyoriented fibers. Mandrel translation speed was set to 18 cm/s. Prior toelectrospinning, the mandrel was wrapped with a small section ofaluminum foil to aid in tube removal. Upon completion ofelectrospinning, the foil liner was slid off the mandrel, and carefullyremoved from the inside of the tube (inner diameter: 5 mm, thickness:˜500 microns, length: 10 cm).

Example 2 Tubular Scaffolds of Bioabsorbable Polymers and CollagenFabricated by Electrospinning Processes 1) Collagen Electrospun Tubes

Collagen (Bovine Collagen Type I, Kensey Nash, Exton, Pa.) waselectrospun at a concentration of 120 mg/ml in HFP. Collagen solutionswere mixed and allowed to sit overnight inside a dark box placed on ashaker plate to ensure that all collagen was dissolved. For collagentubes, a small volume (0.2-0.5 ml depending on mandrel diameter) ofcollagen solution was drawn into a 1 ml Beckton Dickinson syringe andelectrospun onto the rotating mandrel to aid in tube removal. Thispreliminary coating of collagen was dispensed through a blunted 18 gaugeneedle at a rate of 3 ml/hr. The two high voltage power supplies wereconnected to the needle tip and the 5″ diameter back target placed 6″behind the mandrel (2 or 5 mm in diameter), and are set to +25 kv and−10 kv, respectively. The grounded mandrel was placed 8″ from thecharged needle tip, and rotates at a rate of ˜400 rpm to produce ascaffold of randomly oriented fibers. Mandrel translation distance was18 cm, with a translational speed of 18 cm/s. Upon completion of thepreliminary sacrificial layer of collagen, the initial syringe wasdisposed of and a new syringe containing the desired volume of collagensolution was placed on the syringe pump. This solution was electrospunusing the same parameters as the sacrificial layer. Upon completion ofthe electrospinning process, the mandrel was removed from theelectrospinning chamber, and the graft was carefully slid off themandrel. During this process the initial layer was torn away from thegraft, leaving a thin layer of collagen still on the mandrel.

2) PDO and Collagen Electrospun Tubes

50:50 PDO:collagen scaffolds are scaffolds composed of a 50:50 ratio byvolume of 100 mg/ml PDO and 120 mg/ml collagen (Bovine Collagen Type I,Kensey Nash, Exton, Pa.) solutions. The two polymer solutions were madein separate scintillation vials under conditions identical to those ofelectrospinning the polymers individually by placing the polymers in HFPsolution overnight in a dark box on a shaker plate. Once the polymershad completely dissolved equal volumes of the two solutions werecombined together in a new scintillation vial, vortexed for 30 seconds,and placed on a shaker. While the two solutions were mixing, a smallvolume of pure collagen solution is electrospun onto the groundedmandrel to serve as a sacrificial layer using a process identical tothat in the above protocol for electrospinning pure collagen tubes. Oncethe preliminary collagen layer had been electrospun, the desired volumeof blended PDO:collagen solution was drawn into a Beckton Dickinsonsyringe and electrospun onto the rotating mandrel (2 or 5 mm indiameter) to aid in tube removal. Upon completion of the preliminarysacrificial layer of collagen, the initial syringe was disposed of and anew syringe containing the desired volume of collagen solution wasplaced on the syringe pump. This solution was electrospun using the sameparameters as the sacrificial layer and as described in example 2, part1.

3) Cross-Linking Collagen and PDO:Collagen ESS Tubes with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)

Pure collagen scaffolds, as well as blends of PDO and collagen preparedin example 2, parts 2 and 3 were cross-linked using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) inpure ethanol. Samples were soaked in a 40 mM (50× the molarconcentration of collagen in HFP) solution of EDC in ethanol for 18 hrs,followed by a 2 hr rinse in 0.1 M disodium phosphate solution tohydrolyze any unreacted O-isoacylurea intermediates. Aftercross-linking, samples were rinsed in de-ionized water, frozen, andlyophilized overnight to remove any residual moisture.

Example 3 PDO Sheet Scaffolds of Bioabsorbable Polymers Fabricated byElectrospinning Processes

Solutions of 100 mg/ml of PDO were made by placing the polymer in HFPsolvent and leaving the solution overnight in dark box on a shaker plateto ensure that all PDO was dissolved and forms a homogenous solution.The desired volume of polymer solution was then drawn into a plasticBeckton Dickinson syringe and placed in the syringe pump to be dispensedat a rate of 10 ml/hr. Two high voltage power supplies were used. Onewas used to apply a voltage of +20 kV to a blunt tip 18 gauge needlefixed to the solution containing syringe, while the other provides −8 kVto a metal target placed 6″ behind the grounded mandrel (2.5 cm indiameter). The grounded mandrel was placed 8″ from the needle tip androtating at a rate of ˜400 rpm to produce a scaffold of randomlyoriented fibers. Mandrel translation distance was 18 cm, with atranslational speed of 18 cm/s. Immediately after electrospinning themandrel and scaffold are quickly immersed in an ethanol bath to assistin sliding the tube off the mandrel. The tube is cut to form a sheet andthen placed in a fume hood for 30 minutes to allow for the evaporationof any residual ethanol.

Example 4 50:50 PDO:Collagen Sheet Scaffolds of Bioabsorbable Polymersand Collagen Fabricated by Electrospinning Processes

50:50 PDO:collagen scaffolds are scaffolds composed of a 50:50 ratio byvolume of 100 mg/ml PDO and 120 mg/ml collagen solutions and were madeby a process as described in Example 3.

The 50:50 PDS:Collagen sheets were then cross-linked using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) inpure ethanol. Samples were soaked in a 40 mM (50× the molarconcentration of collagen in HFP) solution of EDC in ethanol for 18 hrs,followed by a 2 hr rinse in 0.1 M disodium phosphate solution tohydrolyze any unreacted O-isoacylurea intermediates. Aftercross-linking, samples were rinsed in de-ionized water, frozen, andlyophilized overnight to remove any residual moisture.

Example 5 Tubular Scaffolds of Bioabsorbable Polymers Fabricated by aLyophilization Processes

1) 35/65 poly(caprolactone-co-glycolide) (35/65 PCL/PGA) LyophilizedTubes (10 wt % Solution in 1,4-dioxane)

This example describes the making of a tube containing porous structuresthat would provide pathways for nutrient transport and guided tissueregeneration. Hence, a 10% wt./wt. polymer solution was prepared bydissolving 1 part 35/65 PCL/PGA (Ethicon, Inc., Somerville, N.J.) with 9parts of solvent 1,4-dioxane. The solution was prepared in a flask witha magnetic stir bar. To dissolve the copolymer completely, the mixturewas gently heated to 60° C. and continuously stirred overnight. A clearhomogeneous solution was then obtained by filtering the solution throughan extra coarse filter (Pyrex brand extraction thimble with fritteddisc).

A lyophilizer (DURA-STOP, FTS Systems, Stone Ridge, N.Y.) was used tothen form the tubes from the polymer solution. The freeze dryer waspowered up and the shelf chamber was maintained at −17° C. forapproximately 30 minutes. Thermocouples to monitor the shelf temperaturewere attached for monitoring. The homogeneous polymer solution preparedin Step A was poured into a gap between the barrel of a 1 ml BecktonDickinson (BD) syringe and the plunger of 1 ml BD syringe, thereby usingthe syringe as a mold to form the tube. The mold was placed into thelyophilizer maintained at −17° C. (pre-cooling). The lyophilizationcycle was started and the shelf temperature was held at −17° C. for 15minutes and then, held at −15° C. for 120 minutes. A vacuum was appliedto initiate drying of the dioxane by sublimation. The shelf temperaturewas raised to −5° C. and held at this temperature for 60 minutes. Theshelf temperature was raised to 5° C. and held for 60 minutes. The shelftemperature was raised again to 20° C. and held at that temperature for60 minutes. A second stage of drying was started and the shelftemperature was held at 20° C. for an additional 120 minutes. At the endof the second stage, the lyophilizer was brought to room temperature andatmospheric pressure. The thin scaffold was removed from the plunger ofthe syringe.

2) 35/65 poly(caprolactone-co-glycolide) (35/65 PCL/PGA) LyophilizedTubes (5 wt % Solution in 1,4-dioxane)

This example describes the making of a tube containing porous structuresthat would provide pathways for nutrient transport and guided tissueregeneration. Hence, a 5% wt./wt. polymer solution was prepared bydissolving 1 part 35/65 PCL/PGA with 9 parts of solvent 1,4-dioxane. Thesolution was prepared in a flask with a magnetic stir bar. To dissolvethe copolymer completely, the mixture was gently heated to 60° C. andcontinuously stirred overnight. A clear homogeneous solution was thenobtained by filtering the solution through an extra coarse filter (Pyrexbrand extraction thimble with fitted disc).

A lyophilizer (DURA-STOP, FTS systems, Stone Ridge, N.Y.) was used tothen form the tubes from the polymer solution. The freeze dryer waspowered up and the shelf chamber was maintained at −17° C. forapproximately 30 minutes. Thermocouples to monitor the shelf temperaturewere attached for monitoring. The homogeneous polymer solution preparedin Step A was poured into a gap between the barrel of a 1 ml BD syringeand the plunger of 1 ml BD syringe, thereby using the syringe as a moldto form the tube. The mold was placed into a lyophilizer maintained at−17° C. (pre-cooling). The lyophilization cycle was started and theshelf temperature was held at −17° C. for 15 minutes and then, held at−15° C. for 120 minutes. A vacuum was applied to initiate drying of thedioxane by sublimation. The shelf temperature was raised to −5° C. andheld at this temperature for 60 minutes. The shelf temperature wasraised to 5° C. and held for 60 minutes. The shelf temperature wasraised again to 20° C. and held at that temperature for 60 minutes. Asecond stage of drying was started and the shelf temperature was held at20° C. for an additional 120 minutes. At the end of the second stage,the lyophilizer was brought to room temperature and atmosphericpressure. The thin scaffold was removed from the plunger of the syringe.

Example 6 Preparation of Poly(lactide) and Poly(glycolide) Non-WovenTubes

Nonwoven tubes possessing a length of approximately 50 mm and aninternal diameter of approximately 4 mm and a wall thickness ofapproximately 0.5-1.0 mm were fabricated from various bioresorbablefilaments. Specifically, filaments were comprised of poly(lactide) (PLA)and poly(glycolide) (PGA) and a copolymer of PGA and PLA in a 90:10molar ratio (90/10 PGA/PLA). These samples were fabricated using a drylay nonwoven technique to first produce a non-woven batt from filamentsapproximately 20 microns in diameter and approximately 50 mm in length.This batt was then consolidated via needle punching using a mandrel.

Example 7 Preparation of a Collagen Coated Absorbable, Synthetic TissueEngineered Tubular Scaffold

Highly purified atelocollagen (Colbar, a Johnson & Johnson Co., Israel)is used for coating a tissue engineered tubular scaffold, prepared asdescribed in Examples 1-6 and 8-11. The tissue engineered tubularscaffold (Example 1, part 2) was placed on a mandrel and immersed in 10mM HCl solution containing 1 mg/ml collagen. The tissue engineeredtubular scaffold was soaked in the collagen solution for 30 minutes atroom temperature. The tissue engineered tubular scaffold was removedfrom the solution and dried at room temperature for 8 hours.

Example 8 Preparation of an Acellular Omental Matrix Coated TissueEngineered Tubular Scaffold

Pig omentum is placed in 0.9% saline after harvest. After rinsing in thesaline solution 3 times to rinse off blood and other extraneous debris,the omentum is placed in 70% ethanol for 30 minutes. Following thetreatment with 70% ethanol, the tissue is dehydrated in 100% ethanol for30 minutes with two changes into fresh ethanol. The tissue is thentransferred to acetone for 180 minutes, using fresh solution every 60minutes. Subsequently, the tissue is placed in a 50:50 acetone-hexanemixture for 60 minutes, followed by a 20:80 mixture of the same for24-48 hours (with 3 changes of fresh solution) for lipid removal. Thetissue is then transferred to 100% ethanol for 30 minutes andsubsequently to 70% ethanol where, if necessary, it could be stored at4° C. until the decellularization process is initiated. The tissue isthen immersed in a decellularization buffer comprising TRITON® X-100 (1%w/V; a nonionic detergent) (Sigma-Aldrich, St. Louis, N.J.) and MgCl₂(1%) dissolved in 50 mM Tris-HCl (pH 7.2), for 30 minutes. This isfollowed by treatment in an enzyme solution comprising endonuclease(BENZONASE; 41.8 U/ml, Sigma-Aldrich, St. Louis, N.J.) mixed with thedecellularization buffer. The tissue is spun in this solution for 20hours. The tissue is then washed twice (2 hours each) in a solutioncomprising 50 mM Tris-HCl (pH 7.2), 5 mM MgCl₂ and 1% (W/V) TRITON®X-100. The tissue is then placed in a cell extracting solutioncomprising 1M NaCl, 20 mm EDTA, 0.2% (W/V) TRITON® X-100 pH 7.0 for 1hr, following which the tissue is washed with ultra pure water (4 times,5 minutes each). The tissue is transferred to disinfection solutioncomprising 80:20 water:ethanol (200 proof) with 0.15% peracetic acid (oracetic acid) for 1 hour after washing in water (4 times, 20 minuteseach), the tissue is stored in 70% alcohol at 4° C.

The acellular omentum is air dried and then cryogenically milled into apowder. The powder is then dispersed into 10 mM HCl at a concentrationof 5 mg/ml. A tissue engineered tubular scaffold, as described inExamples 1-6 and 9-11 is placed onto a mandrel and immersed in theacellular omentum suspension and dried by lyophilization at −20° C. for24 hours. The omental matrix on the impregnated tissue engineeredtubular scaffold is then cross linked by thermal dehydration at 120° C.overnight under vacuum.

Example 9 Generation of Cell Sheets on PCL/PGA Films

This example describes the generation of cells sheets comprised of Humanumbilical vein endothelial cells (HUVECs) on films. These sheets canthen be fabricated into tubular structures leading to TEBV comprisedsolely of human umbilical vein endothelial cells (HUVEC). Humanumbilical vein endothelial cells (HUVEC) were seeded onto PCL/PGA filmsto obtain cell sheets. To do this, films were cast by adding 2.5 ml ofthe polymer solution (45/55 PCL/PGA 10% (w/w) in dioxane or 35/65PCL/PGA 10% (w/w) in dioxane) onto a 60 mm culture dishes. Aftercasting, films were sterilized by washing in ethanol and air-dried.HUVEC were harvested by trypsinization and counted using a Guavainstrument (Guava Technologies, Hayward, Calif.). Cells were seeded ontothe films at a density of 5000 cells/cm² (141,350 cells/60 mm dish) andthen placed in a 37° C. incubator for 9 days. Cells were visualized bymicroscopy or by calcein staining The HUVEC cells grew to a confluentlayer on a poly(caprolactone-co-glycolide 35/65 mole-mole %) (35/65PCL/PGA) film or a poly(caprolactone-co-glycolide 45/55 mole-mole %)(45/55 PCL/PGA) film providing a cell sheet. Microscopic imagesconfirmed there was a confluent monolayer of cells. Calcein stainingshowed cells attached and proliferated at days 9 with little to noevidence of dead cells.

The sheets can be rolled into a tube to form a construct that can beused as a tissue engineered blood vessel alone or in combination with amechanical strut such as the scaffolds described in Examples 1-8 or 11.By similar methods (see examples 18 and 19), cell sheets can be formedinto a tube directly.

Example 10 Generation of Cell Sheets on PCL/PGA Films

This example describes the generation of cells sheets comprised of humanumbilical tissue derived cells (hUTCs) on films. Human umbilicaltissue-derived cells are obtained by methods described in U.S. Pat. No.7,510,873 incorporated by reference in its entirety. These sheets arefabricated into tubular structures leading to TEBV comprised solely ofhUTC. hUTCs are seeded onto PCL/PGA films to obtain cell sheets. To dothis, films are cast by adding 2.5 ml of the polymer solution onto a 60mm culture dishes. After casting, films are sterilized by washing inethanol and air-dried. hUTCs are harvested by trypsinization and countedusing a Guava instrument. Cells are seeded onto the films at a densityof 5000 cells/cm² (141,350 cells/60 mm dish) and are then placed in a37° C. incubator. Cells are visualized by microscopy or by calceinstaining Cell sheets comprised of hUTCs and apoly(caprolactone-co-glycolide 35/65 mole-mole %) (35/65 PCL/PGA) filmor a poly(caprolactone-co-glycolide 45/55 mole-mole %) (45/55 PCL/PGA)film are prepared.

The sheets can be rolled into a tube to form a construct that can beused as a tissue engineered blood vessel alone or in combination with amechanical strut such as the scaffolds described in Examples 1-8 or 11.By similar methods (See examples 18 and 19), cell sheets can be formedinto a tube directly.

Example 11 Lyophilized, Decellularized Cell Sheets

This example relates to the use of lyophilized or decellularized cellsheets to fabricate TEBV. The cell sheets will be generated in vitro andthen lyophilized or decellularized. When needed, the required type ofcell sheets can be thawed and then formed into tubular structures toproduce TEBV. Decellularized cell sheets, on the other hand, can bewrapped around other cell sheets to enhance the construction of TEBV byproviding trophic factor support or extracellular matrix proteins.

Cell sheets will be generated as in Examples 9, 10 and 17. Alternativemethods of obtaining cell sheets will include culturing cells ondecellularized omentum (Example 8), on tissue-culture plastic, or onthermoresponsive dishes (CellSeed, Inc, Tokyo, Japan). Cell types usedfor obtaining cell sheets will include endothelial cells, smooth musclecells, skeletal muscle cells, or hUTC. Cells will be maintained inculture until a monolayer is achieved. The resulting cell sheets willthen be processed for vitrification by cryopreservation and subsequentlyophilization (Core Dynamics, Orangeburg, N.Y.).

Example 12 Attachment and Growth of Human Umbilical Tissue-Derived Cellson PDO ESS Scaffolds

This example relates to the use of human umbilical tissue-derived cells(hUTC) to produce tissue-engineered blood vessels (TEBVs). TEBVs can begenerated by seeding vascular grafts or scaffold materials with humanumbilical tissue-derived cells. It is envisioned that seeding hUTC ontothe TEBVs will enhance the seeding, attachment, and proliferation ofendothelian cells (ECs) and smooth muscle cells (SMCs) when seeded invitro or onto the vascular grafts after implantation. hUTC may alsopromote the infiltration and subsequent differentiation of the EC or SMCprogenitor cells into the graft construct. This may promote thematuration and the engraftment of TEBVs during the in vivo implantationby providing trophic support, or providing the expression of ECMproteins.

Attachment and growth of hUTC on PDO and PDO/collagen (50/50,crosslinked) (Examples 1 and 2) ESS scaffolds were assessed. Biopsypunches 5 mm in diameter were made from the scaffold materials andpre-wet in complete growth medium. hUTC were then trypsinized, countedand resuspended at a concentration of 200,000 cells/ml in completegrowth medium. The scaffold punches were placed in 96-well low clusterplates and seeded with 100 microliters of the cell suspension (20,000cells/punch). Cells were allowed to attach for 3 hours at 37° C., andthen the scaffolds were transferred to 24-well low cluster platescontaining 1 ml of complete growth medium. The scaffolds were culturedfor 7 days with a medium change after 3 days.

On day 3 and day 7 post-seeding, the scaffolds were transferred to freshlow cluster 24-well dishes containing 1 ml serum-free DMEM. Thescaffolds were then washed with an additional 1 ml serum-free DMEM. Astock solution of Live/Dead stain (Invitrogen, Carlsbad, Calif.)containing 2 micromolar calcein AM and 4 micromolar ethidum homodimerwas prepared and 0.5 ml was added to each well. After incubation at roomtemperature for 5 minutes, cell attachment and viability of cells wasassessed by fluorescence microscopy.

Results:

The hUTC attached and grew on the TEBV scaffolds. PDO/collagen-ESSscaffolds exhibited more significant increase in the number of cellsfrom day 3 to day 7 as compared to the PDO-ESS scaffolds (FIG. 1).

Example 13 Attachment and Growth of Human Umbilical Artery Smooth MuscleCells (UASMCs) and Human Umbilical Vein Endothelial Cells (HUVECs) onPDO ESS Scaffolds

Attachment and growth of human umbilical artery smooth muscle cells(UASMCs) and human umbilical vein endothelial cells (HUVECs) on PDO ESSscaffolds (100 mg/ml and 140 mg/ml, Example 1) was assessed. UASMCs(Lonza Rockland Inc., Rockland, Me.) and HUVECs (Lonza Rockland Inc.,Rockland, Me.) were seeded onto PDO ESS scaffolds, and at specified timepoints (day 3 and day 7) cells grown on the different surfaces wereassessed for viability by Live/Dead staining.

Sterile PDO scaffolds (5 mm biopsy punches) were placed into empty lowcluster 96-well dishes, washed with PBS, and then soaked in appropriatemedium (EGM-2 for HUVECs, and SmGM for UASMCs) while trypsinizing cells.UASMCs and HUVECs were harvested by trypsinization, counted andresuspended to a final density of 5×10⁵ cells/ml in SmGM (UASMC) orEGM-2 (HUVEC) medium. One hundred microliters (50,000 cells) of thisstock cell suspension was aliquotted onto the scaffolds, and the cellswere allowed to attach for 3 hours in 37° C. incubator. The scaffoldswere then transferred to 24-well dishes containing 1 ml of theappropriate medium and cultured for 3 and 7 days.

The scaffolds were transferred to fresh low cluster 24-well dishescontaining 1 ml serum-free DMEM. The graft materials were then washedwith an additional 1 ml serum-free DMEM. A stock solution of Live/Deadstain containing 2 micromolar calcein AM and 4 micromolar ethidumhomodimer in was prepared serum-free DMEM and 0.5 ml was added to eachwell. After incubation at room temperature for 5 minutes, cellattachment and viability of cells was assessed by fluorescencemicroscopy.

Results:

All samples showed cell attachment and growth over the 7 day cultureperiod (FIGS. 2 and 3). The endothelial cells formed an intact monolayeron the surface of the scaffolds. The 100 mg/ml PDO scaffold exhibitedthe best results, with a high number of attached cells at day 3 andincreases in cell number on day 7. The 140 mg/ml PDO scaffold showed ahigh number of attached cells on day 3, but the increase in cells by day7 was not as dramatic as the 100 mg/ml scaffold.

Example 14 Effect of hUTCs on Proliferation and Migration of HUVECs

This example relates to the use of human umbilical tissue-derived cells(hUTCs) to produce tissue-engineered vascular grafts (TEBVs). TEBVs canbe generated by seeding vascular grafts or scaffold materials with humanendothelial cells (ECs) and human smooth muscle cells (SMCs). It isenvisioned that hUTCs will enhance the seeding, attachment, andproliferation of ECs and SMCs on the vascular grafts. hUTCS may alsopromote the differentiation of the EC or SMC or progenitor cells in thegraft construct. This may promote the maturation of TEBVs during the invitro culture as well as the engraftment during the in vivoimplantation. hUTCs may provide trophic support, or provide and enhancethe expression of ECM proteins.

As proof of principle, the effects of hUTC on the proliferation andmigration of HUVEC are investigated in vitro. For studies ofproliferation, the effects of hUTC lot#120304 were tested and threeendothelial cell types from different vascular beds were used asresponder cells (human umbilical vein endothelial cells[HUVEC], humancoronary artery endothelial cells [HCAEC], and human iliac arteryendothelial cells [HIAEC]). Co-culture with hUTC resulted in enhancedproliferation of endothelial cells. Co-culture with mesenchymal stemcells (MSC) or fibroblasts resulted in cell numbers comparable to mediacontrols (Table 1).

Migration was quantitated by counting the number of cells that were onthe underside of a transwell and both HUVEC and HCAEC were used asresponder cells. Unlike the studies with proliferation, the migratoryresponses of these cells are slightly different. HUTC lot#120304 inducedthe migration of both HUVEC and HCAEC. MSC did not induce the migrationof HUVEC suggesting specificity of this response to hUTC (Table 2).

TABLE 1 Effect of hUTC lot#120304, MSC, and fibroblasts on theproliferation of endothelial cells. Endothelial cells (human umbilicalvein endothelial cells, human iliac artery endothelial cells, humancoronary artery endothelial cells) were seeded onto the bottom of a24-well tissue culture dish at a density of 5000 cells/cm² (10,000cells/well) and hUTC lot#120304, MSC, or fibroblasts inside transwellinserts at a density of 5000 cells/cm² (1,650 cells/insert) inco-culture media (Hayflick 80% + EGM-2MV 20% or Hayflick 50% + EGM-2MV50%). After 7 days of co-culture, cells were harvested and counted usinga Guava instrument. Endothelial cells were also maintained in EGM-2MVmedia as positive control. HUVEC HIAEC HCAEC average std dev average stddev average std dev EGM-2MV 100% 36511.33 1307.32 18100 1609.9413 273283802 Hay80/EGM20 (H80) 6532.33 625.94 8770.6667 187.37752 7391 978 hUTC120304 (H80) 13394.67 2011.56 10961.667 1678.5 12957 445 MSC (H80)5674.33 716.29 9555.6667 933.66286 8136 620 fibroblasts (H80) ND ND 86301049.4805 ND ND Hay50/EGM50 (H50) 6778.5 1175.92 21847.5 2947.9282 7818837 hUTC 120304 (H50) 26595.667 4398.96 24577.333 3421.4854 16056 4225MSC (H50) 5554.67 2801.54 16065 2181.5799 8035 2198 fibroblasts (H50) NDND 12158 2113.0894 ND ND

TABLE 2 Effect of hUTCs and MSCs on the migration of endothelial cells.HUVEC or HCAEC were seeded inside transwell inserts at a density of 5000cells/cm² (23,000 cells/insert) and hUTC lot# 120304 or MSC onto thebottom of a 6-well tissue culture dish at a density of 5000 cells/cm²(48,000 cells/well) in co-culture media (Hayflick 50% + EGM-2MV 50%).After 7 days of co-culture, cells that were on the underside of thetranswell insert were harvested and counted using a Guava instrument.Endothelial cells were also maintained in EGM-2MV media as control.HUVEC HCAEC average std dev average std dev EGM-2MV 3125.67 1849.46848.33 539.13 Hayflick 50% 805.33 323.96 1926.67 280.42 hUTC 1203042402.33 880.1 9071.67 3792.28 MSC 383 124.65 ND ND

Example 15 Preparation of a Cell Lysate-Augmented Biopsy Punches

In this example, hUTC were culture expanded, harvested, lysed byrepeated freeze-thaw cycles, and applied to the bioabsorbable scaffoldsof Examples 1-11 and lyophilized. The cell lysate augmented scaffoldscan be implanted as such or seeded with cells (as in Examples 16, 17) orminced tissue (Examples 25) and cultured to create tissue engineeredblood vessels.

Umbilical Postpartum cells hUTC (Passage 11, lot 120304) were culturedat 5,000 cells per cm squared in T225 cm² cell culture flasks (CORNING,Cat No 431082, Corning, N.Y.) with complete growth media: DMEM-lowglucose (GIBCO, Cat No 11054 Invitrogen, Carlsbad, Calif.), 15% FetalBovine Serum (HyClone Cat No SH30070-03 Logan, Utah) and Pen/Strepsolution (GIBCO, Cat No 15070). After cells expanded to approximately25,000 cells per cm², cells were harvested by TrypLE Select (GIBCO catNo 12563) and collected in 50 ml conical tubes, centrifuged at 300 rcffor 5 minutes and removed the supernatant. The cell pellets were washed3 times with PBS and then re-suspended in PBS at 1×10⁷ cells/ml for atotal volume of 14 ml. This solution was snap frozen in liquid nitrogen,thawed in a 37° C. water bath, centrifuged to remove cell debris, andthe resulting supernatant (10 ml) removed and stored. The remainingmaterial was again snap frozen, thawed, and centrifuged as above. Thesupernatant (2 ml) was removed and stored. The protein concentration ofthe two supernatants was determined by measuring samples diluted in PBSwith the Bradford protein assay kit (BIO-RAD Laboratories Hercules,Calif.). The concentration of the first lysate supernatant (Lot#082908)was 5.2 mg/ml (Lot#082908 low) and the second lysate supernatant was 18mg/ml (Lot#082908 high).

Biopsy punches 5 mm in diameter were taken from PDO-ESS scaffold sheets(Example 3). These scaffolds were placed into 96 well ultra low clusterplate (COSTAR, cat No 3474 Fisher Scientific, Pittsburgh, Pa.), and 25microliters cell lysate was loaded onto each disc at proteinconcentration 5.2 mg/ml (low) or 18 mg/ml (high). The scaffold puncheswere then lyophilized for 48 hours to remove water.

Cell Attachment: The lysate-augmented scaffolds were placed into 96-welllow cluster plates and rehydrated with 25 microliters of EGM-2 medium(Lonza Walkersville, Md.). Human umbilical vein endothelial cells(HUVECs) were tyrpisinized, counted and resuspended to a concentrationof 500,000 cells/ml. Each scaffold was seeded with 100 microliters ofthis cell suspension (50,000 cells) and the cells were allowed to attachfor 3 hours at 37° C. After this attachment period, the scaffolds weretransferred to 24-well low cluster plates containing 1 ml of EGM-2medium. The scaffolds were cultured for 3 and 7 days.

At day 3 and day 7 post-seeding scaffolds were analyzed for cellattachment using Live/Dead stain and for cell number using the CyQuantassay (Invitrogen) to measure cellular DNA. For the Live/Dead stain, thescaffolds were transferred to fresh low cluster 24-well dishescontaining 1 ml serum-free DMEM. The scaffolds were then washed with anadditional 1 ml serum-free DMEM. A stock solution of Live/Dead staincontaining 2 micromolar calcein AM and 4 micromolar ethidum homodimerwas prepared and 0.5 ml was added to each well. After incubation at roomtemperature for 5 minutes, cell attachment and viability of cells wasassessed by fluorescence microscopy.

For the measurement of cellular DNA, the scaffolds were washed in PBS,then frozen in 150 microliters of PBS in microcentrifuge tubes. Thescaffolds were then lyophilized to dryness and resuspended in 150microliters of papain digestion solution. The samples were then digestedovernight at 60° C. The next day, 10 microliters was used to assay forDNA content using the CyQuant NF assay kit (Invitrogen).

Results:

An increase in cell number was observed for both concentrations oflysate tested and at both day 3 and day 7 post-seeding. Live/Deadstaining shows a greater number of cells and more of the scaffoldsurface covered at both timepoints examined (FIG. 4). After 3 days therewas an approximate 4-fold increase in cellular DNA compared to controlscaffolds (FIG. 5).

Example 16 Preparation of Cell Lysate on Tube Scaffolds

PDO tubular scaffolds (Example 1, part 2) were dip-coated into hUTClysate solution (Example 15) at a protein concentration of 5.2 mg/ml(Lot#082908 Low) for 5 minutes then lyophilized for 24 hours using alyophilizer DURA-STOP, FTS system. The freeze dryer was powered up andthe shelf chamber was maintained at −40° C. for approximately 15minutes. Thermocouples to monitor the shelf temperature were attachedfor monitoring. The scaffold tubes were placed into a lyophilizermaintained at −40° C. (pre-cooling). The lyophilization cycle wasstarted and the shelf temperature was held at −40° C. for 15 minutes andthen, held at −37° C. for 60 minutes. A vacuum was applied. The shelftemperature was maintained −40° C. and held at this temperature for 180minutes. The shelf temperature was raised to −25° C. and held for 500minutes. The shelf temperature was raised to −15° C. and held for 180minutes. The shelf temperature was raised to −5° C. and held for 180minutes. The shelf temperature was raised to 5° C. and held for 120minutes. The shelf temperature was held at 20° C. for 120 minutes. Theshelf temperature was held to −20° C. for 120 minutes. Afterlyophilization, the tubular scaffolds were evaluated for cellattachment.

Individual tubular scaffolds were placed into 100 mm untreated plates(from Corning, Cat No 430591). Rat smooth muscle cells were seededstatically onto the tubular scaffolds coated with hUTC lysate orphosphate buffered saline (PBS) (as a control) at a seeding density of5×10⁶/scaffold. Cell seeded scaffolds were incubated in 37° C.humidified air for one hour prior to refeeding the dish with 15milliters smooth muscle growth media. The scaffolds were cultured for 24hours. After 24 hours, the scaffolds were evaluated by Live/Dead kit(from Invitrogen, Cat No L3224). Live/Dead staining on tubular scaffoldscoated with PBS or hUTC lysate and cultured with rat SMC for 24 hoursshowed more cell attached to the lysate-coated scaffold (FIG. 6).

Example 17 Generation of Cell Sheets on the Lysate Treated PDO Sheet

This example is to demonstrate hUTCs seeded on the lysate treated PDOsheet could attach, migrate and penetrate into the scaffold. Thesesheets could be fabricated into tubular structures leading to TEBVcomprised solely of hUTC (see next example) for implantation right awayat the site of injured vessel in vivo or further maturation by culturingin a bioreactor. A PDO sheet (2.0 cm×2.0 cm×0.01 μm) (Lot#3904-78)(Example 3) was soaked with 40 μl lysate (containing 5.2 mg/ml totalprotein, Lot#082908) followed by air drying at 4° C. for overnight. Thetreated scaffold was then seeded with hUTCs, cultured and obtained asdescribed in Example 15, at a density of 1.75×10⁵ cells/cm². The cellseeded scaffold was then cultured in the same condition as for cellsdescribed in Example 15. At 11 and 14 days after seeding, the cellsheets were fixed for H&E staining. As shown, while cells were seeded ononly one side, cells spread all over the surface as well as inside thescaffold, indicating hUTCs attach, migrate and proliferate within thelysate treated PDO scaffold (FIG. 7).

Example 18 Tissue Engineered Graft of Rolled ESS Sheet with hUTC Cellsand hUTC Cell Lysate

Two PDO sheets (2×5×0.05 cm, Lot#5-6-08-2 sheet 3904-50-3) prepared asdescribed in Example 3 were loaded with 700 μl PBS or hUTC lysate(containing 5.2 mg/ml total protein, Lot#082908 Low) and dried out bystoring in −20° C. for 3 days. Each sheet was hydrated with 350 μlgrowth medium (15% FBS in DMEM). 350 μl hUTCs (5×10⁶ cells/ml) wereobtained as described in Example 15 and loaded at a density 1.75×10⁵/cm²(Cell lot#120304, P.9). The cell-loaded sheets were cultured in growthmedium. At day 4, each sheet was cut into two (2×2.5×0.05 cm). One setwas rolled to a single layer tube with 5 mm in diameter and 2.5 cm inlength while the other set stayed in sheet format.

Fibrin sealant (0.5 ml) (Omrix, Biopharmaceutices LTD, Tel Aviv Israel)was applied to the edge of rolled PDO ESS sheet followed by Thrombin(0.5 ml) (Omrix, Biopharmaceutices LTD, Tel Aviv Israel) to glue the endof the scaffold onto itself and keep the scaffold in a tube shape. Theglued rolled tube was removed from the mandrel. A second sheet was keptas a sheet. All tubes and sheets were continued to culture at 37° C. Theviability, attachment and proliferation of hUTCs in tubes and sheetswere evaluated at 1 and 4 days after culture. The results showed thathUTC attached and grew to confluence on PDO sheet with or withoutlysate. Cell attachment is stronger with the lysate treated PDO sheet.While the cell layer formed on PDO sheet is disturbed during a rollingprocess since the cell density was low when evaluated on day 1 after thetube was formed, hUTC uniformly and densely distributed throughout theluminal surface of the tube, with more proliferation found for the PDOmaterial treated with hUTC lysate (FIG. 8).

Example 19 Tissue Engineered Graft of Rolled ESS Sheet with rSMC Cellsand hUTC Cell Lysate

A PDO sheet (2×2.5×0.005 cm, lot#3904-72-23) as described in Example 3was loaded with 60 μl hUTC lysate (containing 5.2 mg/ml total protein,lot#082908 Low). Approximately 250 μl rSMCs (Cell Applications, SanDiego, Calif.) at 3.75×10⁶ cells/ml were seeded on the PDO sheet at adensity of 1.75×10⁵ cells/cm². After 2 hrs at 37° C., the seededmaterial was immersed with SMC growth medium (GM). After 5 days inculture, the sheet was rolled to a tube (˜4 layers) around a mandrel(φ=2 mm).

A laboratory scale machine was fabricated to roll an ESS sheet into agraft that can be used to develop a tissue engineered blood vessel. Themachine has a chuck to which a mandrel was connected that was rotated toallow the sheet to be rolled into a tube. A cell cultured PDO ESS sheetwas placed on the mandrel (5 mm in diameter or 2 mm in diameter). Themandrel was slowly rotated to form 5 layers of a cell containingscaffold. The tube was sealed with fibrin sealant as described inexample 17. The construct was cultured in GM for 5 days and switched toSMC Differentiation Medium (DM) for 4 more days. The construct was cutinto 2 segments, one for Live-dead Staining and the other for H&E byfixing in 10% buffered formalin.

The results show rSMC cells penetrate into the scaffold from both sidesof the scaffold and attach and proliferate well within the scaffoldthroughout the thickness of the material. Some areas (right panel) showthe integration of two layers (FIG. 9).

Example 20 Seeding of hUTC on 2 Mm Diameter Tissue-Engineered BloodVessel Scaffolds

This example relates to the uniform seeding of human umbilical cordcells (hUTC) into PDO-ESS tubes of varying inner diameters, followed byculture to allow cell growth and matrix production. This growth caninclude static culture or culture under physiological conditions in abioreactor, such as luminal flow with or without pressure and/orpulsatile flow. The PDO-ESS tubes can be first coated with type Icollagen or other extracellular matrix component.

PDO-ESS tubes (100 mg/ml, Example 1, part 2) approximately 3.5 cm inlength and 2 mm in inner diameter were coated with collagen by soakingin a solution of 50 micrograms/ml rat tail type I collagen (BDBiosciences, Bedford, Mass.). A collagen-coated tube and a non-coatedcontrol were secured to barbs within a LumeGen bioreactor chamber(Tissue Growth Technologies, Minnetonka Minn.) using silk sutures andthe chambers were sealed. The outer chamber, which bathes the tubularscaffolds in medium, was filled with complete growth medium. hUTC weretrypsinized, counted and resuspended to a concentration of 5.5×10⁵cells/ml in complete growth medium. Seeding rings were then attached tothe LumeGen chamber. These rings allow for rotation of the chamber whenplaced on to a standard tissue culture bottle roller. The cellsuspension was injected into the lumen of the PDO-ESS tubes using asyringe in a way that eliminated all air bubbles. The ends of theluminal chambers were sealed and the chambers were placed on the bottleroller and incubated overnight at 37° C. with a rotation ofapproximately 0.4 rpm. After this overnight incubation, the tubes werecut open and 5 mm biopsy punches taken to examine the distribution ofcells within the TEBV scaffolds. The biopsies of the scaffolds weretransferred to fresh low cluster 24-well dishes containing 1 ml PBS. Astock solution of Live/Dead stain containing 2 micromolar calcein AM and4 micromolar ethidum homodimer was prepared and 0.5 ml was addedseparate wells. The scaffold punches were then transferred to the wellscontaining the Live/Dead solution. After incubation at room temperaturefor 5 minutes, cell attachment and viability of cells was assessed byfluorescence microscopy.

Results:

Profuse cell attachment was observed for all PDO-ESS tubes seeded withhUTC. There was a dramatic increase in the number of cells attached tothe collagen-coated PDO-ESS tube compared to the uncoated PDO-ESS tube.Very few dead cells were observed in either sample (FIG. 10).

Example 21 Bioreactor Processes for Tissue Engineered Blood VesselDevelopment (Static Culture)

This example related to the uniform seeding of smooth muscle cells intoPDO-ESS tubes of varying inner diameters, followed by culture to allowcell growth and matrix production. This growth can include staticculture or culture under physiological conditions in a bioreactor, suchas luminal flow with or without pressure and/or pulsatile flow.

PDO-ESS tubes (100 mg/ml) or PDO/collagen-ESS tubes (Example 1, part 2and Example 2, part 2) approximately 5 cm in length were secured tobarbs within a LumeGen bioreactor chamber (Tissue Growth Technologies,Minnetonka Minn.) using silk sutures. After the chamber was sealed, theouter chamber which bathes the tubular scaffolds was filled with smoothmuscle growth medium (Cell Applications, Inc., San Diego, Calif.). Rataortic smooth muscle cells (Cell Applications, Inc.) were trypsinized,counted and resuspended to a concentration of 2×10⁶ cells/ml in growthmedium. Seeding rings were then attached to the LumeGen chamber. Theserings allow for rotation of the chamber when placed on to a standardtissue culture bottle roller. The cell suspension was injected into thelumen of the PDO-ESS tube using a syringe. The ends of the luminalchamber were sealed and the chamber was placed on the bottle roller andincubated overnight at 37° C. with a rotation of approximately 0.4 rpm.After this overnight incubation, some tubes were cut open and 5 mmbiopsy punches taken to examine the distribution of cells within theTEBV scaffolds. The biopsies of the scaffolds were transferred to freshlow cluster 24-well dishes containing 1 ml serum-free DMEM. Thescaffolds were then washed with an additional 1 ml serum-free DMEM. Astock solution of Live/Dead stain containing 2 micromolar calcein AM and4 micromolar ethidum homodimer was prepared and 0.5 ml was added to eachwell. After incubation at room temperature for 5 minutes, cellattachment and viability of cells was assessed by fluorescencemicroscopy.

Cell-seeded PDO-ESS scaffolds in the LumeGen chambers were thenconnected to the LumeGen bioreactor that is capable of generatingphysiological flow rate, pulsatile flows and pressures. The pulsatileflow comes partly from a peristaltic pump, while the pressure and pulsescan be adjusted by crimping the outlet media flow tubing from either thelumen or chamber. In addition, pulses can optionally be added through amechanism that compresses the graft in a pulsatile manner. This iscontrolled through a computer interface. Flow was initiated and thecells were exposed to a flow rate of 10 ml/min for 2 hours. After thisflow period, one TEBV scaffold was removed form the chamber, andanalyzied using Live/Dead stain as above. Another cell-seeded PDO-ESSscaffold was then cultured statically within the LumeGen chamber for 7days followed by analysis with Live/Dead stain as above.

Results:

The Live/Dead stain results show that the rotating seeding method withinthe LumeGen chamber enables cells to attach and spread on the PDO-ESSscaffolds with a homogeneous distribution of cells throughout the lengthof the scaffold (FIG. 11). Furthermore, the exposure of cells to 10ml/min flow did not shear the cells form the luminal surface of thescaffold. Incubating the seeded scaffold for 7 days following flowresulted in an increase in cell number without affecting the viabilityof the cells (FIG. 12).

Example 22 Bioreactor Processes for Tissue Engineered Blood VesselDevelopment (Pulsatile Flow Physiological Conditions-Short Term Culture)

This example relates to the uniform seeding of smooth muscle cells intoPDO-ESS tubes of varying inner diameters, followed by culture underdynamic conditions to allow cell growth and matrix production. Thesedynamic conditions can include culture under physiological ornon-physiological conditions in a bioreactor, such as luminal flow withor without pressure and/or pulsatile flow. In addition, flow can beintroduced to the outer chamber, which bathes the outside of the tissueengineered blood vessel construct in media.

PDO-ESS tubes (100 mg/ml, Example 1, part 2) approximately 5 cm inlength and 4 mm in diameter were secured to barbs within a LumeGenbioreactor chamber (Tissue Growth Technologies, Minnetonka Minn.) usingsilk sutures and the chambers were sealed. The outer chambers, whichbathe the tubular scaffolds in medium, were filled with smooth musclegrowth medium (Cell Applications, Inc). Rat aortic smooth muscle cells(Cell Applications, Inc.) were trypsinized, counted and resuspended to aconcentration of 2×10⁶ cells/ml in growth medium. Seeding rings werethen attached to the LumeGen chamber. These rings allow for rotation ofthe chamber when placed on to a standard tissue culture bottle roller.The cell suspension was injected into the lumen of the PDO-ESS tubeusing a syringe. The ends of the luminal chamber were sealed and thechamber was placed on the bottle roller and incubated overnight at 37°C. with a rotation of approximately 0.4 rpm.

One cell-seeded PDO-ESS scaffold in the LumeGen chamber was thenconnected to the LumeGen bioreactor that is capable of generatingphysiological flow conditions, including pulsatile flows and pressures.The pulsatile flow comes partly from a peristaltic pump, while thepressure and pulses can be adjusted by crimping the outlet media flowtubing from either the lumen or chamber. In addition, pulses can beadded through a mechanism that compresses the graft in a pulsatilemanner. This is controlled through a computer interface. Flow wasinitiated and the cells seeded on the tubular scaffold were exposed to aflow rate of 20 ml/min and a pulsatile pressure of 120-80 mm Hg at afrequency of 1 Hz. As a control, another cell-seeded tube in thebioreactor chamber was cultured statically with a luminal media changeafter 24 hours.

After three days of culture, the tubular scaffolds were removed from thechambers, cut open, and 5 mm biopsy punches taken to examine the number,distribution, and morphology of cells within the TEBV scaffolds. Thebiopsy punches of the scaffolds were transferred to fresh low cluster24-well dishes containing 1 ml PBS. A stock solution of Live/Dead staincontaining 2 micromolar calcein AM and 4 micromolar ethidum homodimerwas prepared and 0.5 ml was added to separate wells. The scaffoldpunches were then transferred to the wells containing the Live/Deadsolution. After incubation at room temperature for 5 minutes, cellattachment and viability of cells was assessed by fluorescencemicroscopy.

Results:

The Live/Dead stain results show that the rotating seeding method withinthe LumeGen chamber enables cells to attach and spread on the PDO-ESSscaffolds with a homogeneous distribution of cells throughout the lengthof the scaffold. Furthermore, the exposure of cells to 20 ml/min flowand physiological 120-80 mm Hg pulsatile pressure resulted in a dramaticincrease in the number of cells on the surface of the scaffolds. Inaddition, the morphology of the cells was altered to align in thedirection of the flow (FIG. 13).

Example 23 Bioreactor Processes for Cell Seeded Tissue Engineered BloodVessel Development (Pulsatile Flow Physiological Condtions-Long TermCulture)

Cells (umbilical artery smooth muscle cells—UASMCs) are seeded on ascaffold as described in Examples 1-11 in a perfused rotating wallvessel bioreactor (Synthecon, Inc., Houston Tex.). The bioreactor has acentral rotating core with barbs that the scaffolds will be connectedto. The core allows for medium to be perfused through the lumen of thescaffold, while the exterior is bathed in medium. The entire assembly isrotated horizontally as above, again minimizing shear stress. Forseeding of cells onto the luminal surface of the scaffolds, a cellsuspension (10⁶ cells/ml) is pumped into the lumen of the scaffold. Theflow is stopped and rotation continues. As the bioreactor rotates, cellsare attached to the surface of the rotating lumen in a uniform manner.After the 2 to 4 hour incubation period for attachment, the remainingunattached cells are flushed from the lumen, and growth medium perfusedthrough the lumen. This culture period is continued for ˜14 days,allowing the cells to grow and migrate into the pores of the scaffold(the actual time of culture will be determined empirically).

Growth of cells into the scaffolds will be examined by confocalmicroscopy following calcein staining and or actin staining usingrhodamine-phalloidin. Similar techniques would be used to determine thedepth of cell growth and/or migration into scaffold biomaterials.Cross-sectional images will be examined and the depth of cell ingrowthwill be measured using image analysis tools.

Other cell-seeded scaffolds can be transferred to a second bioreactor(Tissue Growth Technologies, Inc.) that is capable of generatingphysiological flow rate, pulsatile flows and pressures. The pulsatileflow comes partly from a peristaltic pump, while the pressure and pulsescan be adjusted by crimping the outlet media flow tubing from either thelumen or chamber. In addition, pulses can be added through a mechanismthat compresses the graft in a pulsatile manner. This is controlledthrough a computer interface. The bioreactor has built-in pressuresensors, as well as a laser micrometer that measures the graft outerdiameter. Flow rate, pressure and the graft outer diameter are graphedon the computer in real time. This allows the user to applyphysiological pressures and pulsatile waves to a graft and to alter themin any way.

The cell-seeded grafts are cultured in this bioreactor for an additionalperiod of time dependent on the desired physical and biologicalcharacteristics desired. During the first period of time, the pressureand flow rate are slowly increased to eventually reached the desiredphysiological levels. The final parameters depend on the eventuallocation of the scaffold. These levels are maintained during the finalstages. For 1 hour each day, the following measurements will berecorded: pressure fluctuation within the bioreactor chamber; flow rate;and fluctuation in the outer diameter of the scaffold. At the end of theculture period, the pressure may be increased until graft failure todetermine the burst-strength of construct.

Example 24 Bioreactor Processes for Cell Sheet Tissue Engineered BloodVessel Development

Cell sheets rolled into a tube as described in Examples 17-18 can befurther bioprocessed to form a tissue engineered blood vessel usingbioreactor processes. The cell sheets will be seeded with cells such ashUTCs or IMAs in a perfused rotating wall vessel bioreactor (Synthecon,Inc.). The bioreactor has a central rotating core with barbs that thescaffolds will be connected to. The core allows for medium to beperfused through the lumen of the scaffold, while the exterior is bathedin medium. The entire assembly is rotated horizontally as above, againminimizing shear stress. For seeding of cells onto the luminal surfaceof the cell sheets, a cell suspension (10⁶ cells/ml) is pumped into thelumen of the tube. The flow is stopped and rotation continues. As thebioreactor rotates, cells are attach to the surface of the rotatinglumen in a uniform manner. After the 2 to 4 hour incubation period forattachment, the remaining unattached cells are flushed from the lumen,and growth medium perfused through the lumen. This culture period iscontinued for ˜14 days, allowing the cells to grow and migrate into thepores of the scaffold (the actual time of culture will be determinedempirically).

Growth of cells into the scaffolds will be examined by confocalmicroscopy following calcein staining and or actin staining usingrhodamine-phalloidin. Similar techniques would be used to determine thedepth of cell growth and/or migration into scaffold biomaterials.Cross-sectional images will be examined and the depth of cell ingrowthwill be measured using image analysis tools.

The cell-seeded scaffolds can be transferred to a second bioreactor(Tissue Growth Technologies, Inc.) that is capable of generatingphysiological flow rate, pulsatile flows and pressures. The pulsatileflow comes partly from a peristaltic pump, while the pressure and pulsescan be adjusted by crimping the outlet media flow tubing from either thelumen or chamber. In addition, pulses can be added through a mechanismthat compresses the graft in a pulsatile manner. This is controlledthrough a computer interface. The bioreactor has built-in pressuresensors, as well as a laser micrometer that measures the graft outerdiameter. Flow rate, pressure and the graft outer diameter are graphedon the computer in real time. This allows the user to applyphysiological pressures and pulsatile waves to a graft and to alter themin any way.

The cell-seeded grafts are cultured in this bioreactor for an additionalperiod of time dependent on the desired physical and biologicalcharacteristics desired. During the first period of time, the pressureand flow rate are slowly increased to eventually reached the desiredphysiological levels. The final parameters depend on the eventuallocation of the scaffold. These levels are maintained during the finalstages. For 1 hour each day, the following measurements will berecorded: pressure fluctuation within the bioreactor chamber; flow rate;and fluctuation in the outer diameter of the scaffold. At the end of theculture period, the pressure may be increased until graft failure todetermine the burst-strength of construct.

Example 25 Preparation of Minced Tissue on Tubular Construct

Two small biopsy tissues were harvested from rat muscle (Lewis rat fromHarlan, Indianapolis, Ind.) by 5 mm diameter biopsy punch (Miltex, REFNo 33-35). Each biopsy weighed around 50-60 mg and was placed in PBSsupplemented with penicillin at standard concentrations (100 U/ml). Thetissue was rinsed three times in PBS and minced into small pieces.Minced tissue was weighed and divided into 25 mg and 50 mg and thenspread evenly on the outer surface of the each tubular construct (FIG.14). The tissue fragments were held on the scaffold by using fibrin glue(EVICEL, Cat No 3905, Ethicon, Somerville, N.J.). The tubular constructwas loaded with minced tissue and placed in an incubator at 37° C. for 2hours and 72 hours (FIG. 15).

Example 26 Preparation of A Minced Tissue-Seeded TEBV Construct With TwoSources of Minced Tissue

This example describes the preparation of a tissue-engineered bloodvessel created from a bioabsorbable scaffold seeded with mincedautologous tissue as the cell source. A tubular or flat bioresorbablescaffold with dimensions as outlined in Examples 1-11 is prepared. Asmall biopsy of tissue containing smooth muscle cells from tissue sourcee.g. muscle layers in the walls of hollow organs (such as the digestivetract, lower part of the esophagus, stomach and intestines, the walls ofthe bladder, the uterus, various ducts of glands and the walls of bloodvessels) is obtained. The biopsied tissue is placed in PBS supplementedwith penicillin at standard concentrations (100 U/ml). The tissue isrinsed three times in PBS and then minced with the help of scalpels toobtain minced tissue. The tissue is then distributed evenly on the outersurface of the tubular or flat construct. Another tissue biopsy isobtained from an endothelial tissue source for e.g. the lining of theblood vessel. The tissue is placed in PBS supplemented with penicillinat standard concentrations (100 U/ml). The tissue is minced anddistributed on the inner surface of the tubular construct or the innersurface of the flat construct. The tissue fragments in both cases can beheld on the scaffold by using cell friendly glues for e.g. fibrin glue.Scaffold constructs that are flat can now be sutured into tubularconstructs. The constructs can then be cultured in medium containingDMEM with Pen/Strep and 15% FBS for 4 to 8 weeks in low cell attachmentdishes, during which smooth muscle cells and endothelial cells willmigrate from the minced tissue on to the scaffolds. The engineeredvessel can then be further cultured in a bioreactor for several weeks ormonths in an atmosphere of 10% CO₂ at a temperature of 37° C. in DMEMsupplemented with 20% FBS, penicillin G (100 U/ml), 5 mM HEPES, ascorbicacid (0.05 mg/ml), CuSO₄ (3 ng/ml), proline (0.05 mg/ml), alanine (0.03mg/ml), and glycine (0.05 mg/ml) as described in Example 27.

Example 27 Preparation of a Minced Tissue-Seeded TEBV Construct with aSingle Source of Minced Tissue

In another example, constructs seeded with minced tissue can be preparedas outlined in Example 25. The source of minced tissue can, however, bea single source such that the single tissue source contains both thesmooth muscle cells and endothelial cells and the same minced tissuefragments are applied to the inner and outer surface of the scaffolds.

Example 28 Bioreactor Processes for Cell Lysate or Minced TissueEngineered Blood Vessel Development

Constructs containing minced tissue or cell lysate and scaffolds asdescribed in Examples 1-11, 24-26 can be further bioprocessed to form atissue engineered blood vessel using bioreactor processes. Theconstructs containing minced tissue and scaffolds will be seeded withcells such as hUTCs or IMAs in a perfused rotating wall vesselbioreactor (Synthecon Inc., Houston Tex.). The bioreactor has a centralrotating core with barbs that the scaffolds will be connected to. Thecore allows for medium to be perfused through the lumen of the scaffold,while the exterior is bathed in medium. The entire assembly is rotatedhorizontally as above, again minimizing shear stress. For seeding ofcells onto the luminal surface of the constructs containing mincedtissue and scaffolds, a cell suspension (10⁶ cells/ml) is pumped intothe lumen of the construct. The flow is stopped and rotation continues.As the bioreactor rotates, cells are attach to the surface of therotating lumen in a uniform manner. After the 2 to 4 hour incubationperiod for attachment, the remaining unattached cells are flushed fromthe lumen, and growth medium perfused through the lumen. This cultureperiod is continued for ˜14 days, allowing the cells to grow and migrateinto the pores of the scaffold (the actual time of culture will bedetermined empirically).

Growth of cells into the scaffolds will be examined by confocalmicroscopy following calcein staining and or actin staining usingrhodamine-phalloidin. Similar techniques would be used to determine thedepth of cell growth and/or migration into scaffold biomaterials.Cross-sectional images will be examined and the depth of cell ingrowthwill be measured using image analysis tools.

The cell-seeded scaffolds can be transferred to a second bioreactor(Tissue Growth Technologies, Inc.) that is capable of generatingphysiological flow rate, pulsatile flows and pressures. The pulsatileflow comes partly from a peristaltic pump, while the pressure and pulsescan be adjusted by crimping the outlet media flow tubing from either thelumen or chamber. In addition, pulses can be added through a mechanismthat compresses the graft in a pulsatile manner. This is controlledthrough a computer interface. The bioreactor has built-in pressuresensors, as well as a laser micrometer that measures the graft outerdiameter. Flow rate, pressure and the graft outer diameter are graphedon the computer in real time. This allows the user to applyphysiological pressures and pulsatile waves to a graft and to alter themin any way.

The cell-seeded grafts are cultured in this bioreactor for an additional7 days. During the first 3 days, the pressure and flow rate are slowlyincreased to eventually reached the desired physiological levels. Thefinal parameters depend on the eventual location of the scaffold. Theselevels are maintained for the final 4 days. For 1 hour each day, thefollowing measurements will be recorded: pressure fluctuation within thebioreactor chamber; flow rate; and fluctuation in the outer diameter ofthe scaffold. At the end of the culture period, the pressure may beincreased until graft failure to determine the burst-strength ofconstruct.

Example 29 In-Vivo Efficacy Study of TEBV

TEBVs are surgically implanted in the femoral arteries of 14 adult dogs.5 to 10 mm sections of the native vessel are removed and replaced withthe experimental TEBV using standard surgical techniques. Anastomosesare performed using standard suture techniques. The vessel lumen areirrigated with a standard heparin solution. The muscle and skin areclosed by standard techniques. Postoperatively, the patency is monitoredby standard Ultrasound.

The TEBVs are explanted after 4 weeks and the patency is assessed bydirect inspection. Patency is confirmed by excising the TEBV andevaluating the lumen histologically.

Example 30 Use of TEBV in the Treatment of Coronary Heart DiseasePatient

In a coronary by-pass (CABG) procedure, a TEBV would be used forpatients suffering from arteriosclerosis, a common arterial disordercharacterized by arterial walls that have thickened (blockages), havelost elasticity, and have calcified. This leads to a decrease in bloodsupply which can lead to damage to the heart, stroke and heart attacks.

Thus, a PDO tubular scaffold fabricated by electrospinning processesdescribed in Example 1 and then cell seeded and bioreactor processed asdescribed in Example 10, forms a TEBV that is then sterilized, packagedand delivered to an operating room. In a typical CABG procedure, thesurgeon opens the chest via a sternotomy. The heart's functions aretaken over by a Heart and Lung machine. The diseased artery is locatedand one end of the TEBV is sewn onto the coronary arteries beyond theblockages and the other end is attached to the aorta. The heart isrestarted, the sternum is wired together and the incisions are suturedclosed. Within a few weeks, the successful by-pass procedure is fullyhealed and the patient is functioning normally.

The above description is merely illustrative and should not be construedto capture all consideration in decisions regarding the optimization ofthe design and material orientation. Although shown and described iswhat is believed to be the most practical and preferred embodiments, itis apparent that departures from specific designs and methods describedand shown will suggest themselves to those skilled in the art and may beused without departing from the spirit and scope of the invention. Thepresent invention is not restricted to the particular embodimentsdescribed and illustrated, but should be constructed to cohere with allmodifications that may fall within the scope for the appended claims.

Example 31 Mammary Artery Derived Cells: Isolation and CharacterizationMammary Artery Derived Cell Isolation

Internal mammary artery (IMA) will be obtained from the National DiseaseResearch Interchange (NDRI, Philadelphia, Pa.). To remove blood anddebris, the artery will be trimmed and washed in Dulbecco's modifiedEagles medium (DMEM-low glucose; Invitrogen, Carlsbad, Calif.) orphosphate buffered saline (PBS; Invitrogen). The artery will then bemechanically dissociated in tissue culture plates until the tissue isminced to a fine pulp. The tissue will then be transferred to a50-milliliter conical tube. The tissue will then be digested in anenzyme mixture containing 0.25 Units/milliliter collagenase (ServaElectrophoresis, Heidelberg, Germany), 2.5 Units/milliliter dispase(Roche Diagnostics Corporation, Indianapolis Ind.) and 1Units/milliliter hyaluronidase (Vitrase, ISTA Pharmaceuticals, Irvine,Calif.). The enzyme mixture will then be combined with growth medium(DMEM-low glucose (Gibco), penicillin (50 Units/milliliter) andstreptomycin (50 ug/mL, Gibco)) containing 1% fetal bovine serum (FBS).The conical tube containing the tissue, medium and digestion enzymeswill be incubated at 37° C. in an orbital shaker at 225 rpm for 2 hours.

The digest is centrifuged at 150×g for 5 minutes, the supernatant willthen be aspirated. The pellet will then be resuspended in 20 millilitersof medium. The cell suspension will then be filtered through a 40-micronnylon BD FALCON Cell strainer (BD Biosciences, San Jose, Calif.). Thefiltrate will then be resuspended in medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatantwill then be aspirated and the cells will be resuspended in another 50milliliters of fresh culture medium. This washing procedure will berepeated twice more.

After the final centrifugation, cells will be plated in growth mediumcontaining either 1% FBS or 10% FBS and cultured at 37° C. and 5% CO₂).Fragments of IMA will also be cultured as explants in coated ornon-coated tissue culture flasks. Cells that migrate out of the tissuefragments, under media selection, will be harvested using trypsin orother non-enzymatic methods.

For karyotype analysis, passage 4 and passage 10 mammary artery derivedcells will be plated into T25 flasks and allowed to attach overnight.Flasks will then be filled with REGM and sent to the University ofMedicine and Dentistry of New Jersey for karyotype analysis.

Analysis of Growth Potential

IMA derived cells will be plated at 5000 cells/cm² onto T75 flasks ingrowth medium and cultured at 37° C. in 5% carbon dioxide. Cells will bepassaged every 3-5 days. At each passage, cells are counted andviability is measured using a Guava instrument (Guava Technologies,Hayward, Calif.). Population doublings [ln(final cell yield/initialnumber of cells plated)/ln 2] are then calculated.

Flow Cytometry

Flow cytometry analysis will be performed on IMA derived cells. Cellswill be expanded to passage four and ten in growth medium on T225 flasksat 37° C. and 5% carbon dioxide. Adherent cells will be washed in PBSand detached with Trypsin/EDTA (Gibco). Cells will be harvested,centrifuged and resuspended in 3% (v/v) FBS in PBS at a concentration of1×10⁷ cells/mL. The specific antibody will be added to 100 microlitersof cell suspension and the mixture is incubated in the dark for 30-45minutes at 4° C. After incubation, cells will be washed with PBS andcentrifuged to remove excess antibody. Cells will be resuspended in 500microliters PBS and analyzed by flow cytometry. Flow cytometry analysiswill be performed with a Guava instrument. Antibodies to be used areshown in Table 3.

TABLE 3 Antibodies to be used in characterizing cell surface markers ofIMA derived cells. Antibody Manufacture Catalog number CD34 BDPharmingen 555821 CD44 BD Pharmingen 555478 CD45R BD Pharmingen 555489CD117 BD Pharmingen 340529 CD141 BD Pharmingen 559781 CD31 BD Pharmingen555446 CD133 Miltenyi Biotech 120-001-243 SSEA4 R&D Systems FAB1435PCD105 SantaCruz Biotech SC-21787 CD104 BD Pharmingen 555720 CD166 BDPharmingen 559263 CD29 BD Pharmingen 555442 IgG-FITC BD Pharmingen555748 IgG-PE BD Pharmingen 555749

Total RNA Isolation

RNA will be extracted from IMA derived cells. (RNeasy Mini Kit; Qiagen,Valencia, Calif.). RNA will be eluted with 50 μL DEPC-treated water andstored at −80° C.

Reverse Transcription

RNA will be reversed transcribed using random hexamers with the TaqManreverse transcription reagents (Applied Biosystems, Foster City, Calif.)at 25° C. for 10 minutes, 37° C. for 60 minutes and 95° C. for 10minutes. Samples will be stored at −20° C. Selected genes (see tablebelow) will be investigated using conventional PCR.

PCR

PCR reactions (with the exception of GAPDH—see chart below) will beperformed on cDNA samples using RT² PCR Primer sets (SuperArrayBiosciences Corp, Frederick Md.). All primers shown below will besequence verified.

CATALOG GENE NUMBER Oct 4 PPH02394A Rex 1 PPH02395A Sox2 PPH02471A HumanTERT (hTERT) PPH00995A FGF4 PPH00356A

Primers will be mixed with 1 μL of cDNA and 2× ReactionReady™ SYBR GreenPCR Master Mix (SuperArray Biosciences) according to manufacturer'sinstructions and PCR will be performed using an ABI Prism 7000 system(Applied Biosystems, Foster City, Calif.). Thermal cycle conditions willbe initially 50° C. for 2 min and 95° C. for 10 min followed by 34cycles of 95° C. for 15 sec and 60° C. for 1 min. For GAPDH, PCR will beperformed using GAPDH primers from Applied Biosystems (cat#: 402869) 1μL of cDNA solution and 1× AmpliTaq Gold universal mix PCR reactionbuffer (Applied Biosystems, Foster City, Calif.) according tomanufacturer's protocol. Primer concentration in the final PCR reactionwill be 0.5 μM for both the forward and reverse primer and the TaqManprobe is not added. Samples will be run on 2% (w/v) agarose gel andstained with ethidium bromide (Sigma, St. Louis, Mo.). Images will becaptured using a 667 Universal Twinpack film (VWR International, SouthPlainfield, N.J.) using a focal-length Polaroid™ camera (VWRInternational, South Plainfield, N.J.).

ELISA

IMA derived cells will be thawed at passage four and passage ten andseeded onto T75 flasks at 5000 cells/cm² each containing 15 millilitersof growth medium. Cells will be cultured for 24 hours at 37° C. in 5%carbon dioxide and atmospheric oxygen. The medium will be changed to aserum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v) bovine serumalbumin (Sigma), penicillin (50 Units/milliliter) and streptomycin (50ug/mL, Gibco)) and further cultured for 8 hours. Conditioned, serum-freemedium is then collected at the end of incubation by centrifugation at14,000×g for 5 min and stored at −20° C.

To estimate the number of cells in each flask, cells will be washed withPBS, detached using 2 milliliters trypsin/EDTA (Gibco) and counted witha Guava instrument (Guava Technologies Hayward, Calif.). Samples arethen assayed for the following factors: tissue inhibitor ofmetalloproteinase-1 (TIMP1), tissue inhibitor of metalloproteinase-2(TIMP2), platelet-derived epithelial growth factor bb (PDGFbb),keratinocyte growth factor (KGF), hepatocyte growth factor (HGF),fibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), Heparin-binding epidermal growth factor (HB-EGF), monocytechemotactic protein-1 (MCP1), interleukin-6 (IL6), interleukin-8 (IL8),transforming growth factor alpha (TGFa), brain-derived neurotrophicfactor (BDNF), stromal-derived factor 1B (SDF1B), cilliary neurotrophicfactor (CNTF), basic nerve growth factor (bNGF), neurotrophin-3 (NT3)with the Searchlight Proteome Arrays (Pierce Biotechnology Inc.).

Example 32 Seeding of Rat Smooth Muscle Cells R354-05 into PDO scaffolds

This example relates to the uniform static seeding of rat smooth musclecells R354-05 (Cell Applications) into PDO-ESS tube (Example 1, part 2).Cells are cultured to allow cell growth and matrix production. Thisgrowth can include static culture or culture under physiologicalconditions in a bioreactor, such as luminal flow with or withoutpressure and/or pulsatile flow.

PDO tubes were cut in 2 cm length and placed in a 60 mm tissue culturedish. Rat aortic smooth muscle cells (Cell Applications, Inc.) weretrypsinized, counted and resuspended in rat smooth muscle growth mediumat a concentration of 2×10⁶ cells/ml. Using a 200 ul pipet tip cellswere gently dripped onto the PDO tube. The PDO tubes containing thecells were left at room temperature for one hour before being placed ina 37° C. humidified environment for 4 days.

After four days of static culture PDO tubes were placed in a rotary cellculture system (Synthecon) for ten days at 7.0 rpm to allow cell growthand matrix product. At 14 days in culture, the distribution andmorphology of the cells is evaluated. PDO tubes were transferred to a 60mm tissue culture dish. 5 mm biopsy punches were harvested and placed ina 24 well plate containing PBS. To test the viability of the cells, alive/dead assay (Molecular Probes) was performed. Ten mls of PBScontaining 2 micromolar calcein AM and 4 micromolar ethidum homodimerwere prepared. One ml of the live/dead stain was added to the biopsies.After incubation at room temperature for 5 minutes, cell attachment andviability of cells was assessed by fluorescence microscopy. Similarpunch biopsies were fixed in 10% neutral buffered formalin, embedded inparaffin, sectioned and stained with H&E to evaluate cell morphology(FIG. 16) and Masson's Trichome stain which is specific for theformation of extracellular matrix.

Results:

Rat smooth muscle cells are viable throughout the culture period withthe PDO tubes supporting cell viability. Static seeding of the ratsmooth muscle cells for four days leads to an even cell distribution.Cells attach on both sides of the scaffolds. Cell infiltration into thescaffolds is observed.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.A method of making a tissue engineered blood vessel comprising the stepsof: a. Providing a tubular shaped scaffold comprising poly(p-dioxanone);b. Applying said scaffold with minced muscle tissue and mincedendothelial tissue; and c. Culturing the minced tissue scaffold in abioreactor.
 7. (canceled)
 8. (canceled)
 9. (canceled)